Methods for RNA extraction and detection from a urine sample

Abstract

Provided herein are methods of RNA extraction from a urine sample, detection of cDNA reverse transcribed from extracted RNA and applications thereof (e.g., for the prognosis and / or diagnosis of disease, such as cancer).

Description

METHODS FOR RNA EXTRACTION AND DETECTION FROM A URINE SAMPLECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority from U.S. Provisional Application No. 63 / 678,458, filed August 1, 2024, and U.S. Provisional Application No. 63 / 814,351, filed May 29, 2025, the disclosure of each of which is incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] Provided herein are methods for RNA extraction from a urine sample, detection of cDNA reverse transcribed from extracted RNA and applications thereof (e.g., for the prognosis and / or diagnosis of disease, such as cancer).REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (LXDX_005_02WO_SeqList_ST26.xml;Size: 529,382 bytes; and Date of Creation: July 30, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] Liquid biopsy is a minimally invasive diagnostic approach that allows for the detection and analysis of biomarkers from bodily fluids, providing valuable information for disease diagnosis, prognosis, and / or treatment monitoring. Among various types of biological fluids, urine is an attractive specimen for liquid biopsy due to its ease of collection and non-invasive nature.

[0005] Current methods for RNA extraction from urine face several challenges that limit their efficiency and reliability. Urine contains low concentrations of RNA, which is often degraded or present in fragmented forms. Additionally, the complex composition of urine, which includes proteins and salts, can interfere with RNA extraction and subsequent analyses. Existing RNA extraction techniques often result in low yield and poor quality of RNA, which hampers the sensitivity and accuracy of downstream molecular assays, such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Currently, achieving detectable levels of RNA in urine samples from men for analysis related to prostate cancer, such as prognosis and / or diagnosis, requires an invasive and uncomfortable digital rectal examination (DRE) shortly before urine sample collection.

[0006] Thus, there exists a need for methods for improved extraction and detection of RNA from urine samples, particularly methods that provide an enhanced yield of extracted RNA while minimizing invasiveness and discomfort.SUMMARY OF THE INVENTION

[0007] Provided herein are methods for extracting RNA present in whole urine from a subject’s urine sample, comprising: admixing the whole urine and a first solid support to provide an RNA-bound first solid support, wherein the first solid support comprises silicon carbide; admixing the RNA-bound first solid support and a polar organic solvent to provide an RNA- bound first solid support admixture and performing centrifugation of the RNA-bound first solid support admixture to provide a pellet and a supernatant; removing the supernatant, and washing the pellet with a wash buffer to provide a washed RNA-bound first solid support; admixing wash buffer, the washed RNA-bound first solid support, and a second solid support to provide an RNA-bound second solid support, wherein the second solid support comprises a silicon-based compound; washing the RNA-bound second solid support to provide a washed RNA-bound second solid support; treating the washed RNA-bound second solid support with deoxyribonuclease (DNase) to provide a DNase-treated second solid support and a flowthrough; washing the DNase-treated second solid support to provide a washed DNase-treated second solid support; drying the washed DNase-treated second solid support to provide a dried second solid support; and eluting RNA from the dried second solid support to provide extracted RNA.Also provided herein are compositions comprising whole urine and a solid support comprising silicon carbide.Further provided herein are compositions comprising whole urine and an RNA-bound solid support.Further provided herein are methods for making an RNA-bound solid support, comprising admixing whole urine and a solid support comprising silicon carbide to provide the RNA-bound solid support.Further provided herein are methods for making a plurality of RNA-bound solid supports, comprising admixing whole urine and a plurality of solid supports comprising silicon carbide to provide the plurality of RNA-bound solid supports.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 is a graph showing the number of qPCR cycles (Crt) used to generate a detectable amount of cDNA reverse transcribed from KLK3 RNA extracted according to the method of Example la, as compared to cDNA reverse transcribed from RNA extracted using a Thermo Fisher RNA extraction method. S#### identifies individual samples from 24 men.DETAILED DESCRIPTION

[0009] Provided herein are methods for extracting RNA present in whole urine from a subject’s urine sample, comprising: admixing the whole urine and a first solid support to provide an RNA-bound first solid support, wherein the first solid support comprises silicon carbide; admixing the RNA-bound first solid support and a polar organic solvent to provide an RNA- bound first solid support admixture and performing centrifugation of the RNA-bound first solid support admixture to provide a pellet and a supernatant; removing the supernatant, and admixing the pellet and wash buffer to provide a washed RNA-bound first solid support; admixing wash buffer, the washed RNA-bound first solid support, and a second solid support to provide an RNA-bound second solid support, wherein the second solid support comprises a silicon-based compound; washing the RNA-bound second solid support to provide a washed RNA-bound second solid support; treating the washed RNA-bound second solid support with DNase to provide a DNase-treated second solid support and a flowthrough; washing the DNase-treated second solid support to provide a washed DNase-treated second solid support; drying the washed DNase-treated second solid support to provide a dried second solid support; and eluting RNA from the dried second solid support to provide extracted RNA.

[0010] The methods provided herein result in significantly improved RNA extraction and detection, e.g., by reverse transcription (RT) and qPCR, such that urine samples that would otherwise provide from standard methods (e.g., the Thermo Fisher method or Norgen method described herein) an amount of RNA that is insufficient for downstream molecular analyses can now provide an at least sufficient quantity of RNA for the same molecular analyses.

[0011] In some embodiments, extracted RNA is provided in an amount that is at least 3 -fold greater than RNA obtained from a standard RNA extraction method.DEFINITIONS

[0012] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

[0013] As used herein, the term “subject” includes, but is not limited to, a mammal (e.g., a human, a non-human primate, a murine, a simian, an equine, a bovine, a porcine, a canine, a feline, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a non- human primate. In some embodiments, the subject is a human subject. In some embodiments, the subject is female. In some embodiments, the subject is a male human subject who has a prostate.

[0014] As used herein, “GG >2 prostate cancer” means Grade Group >2 prostate cancer”. In some embodiments, the GG >2 prostate cancer is GG >3 prostate cancer. In some embodiments, the GG >2 prostate cancer is GG >4 prostate cancer. In some embodiments, the GG >2 prostate cancer is GG5 prostate cancer.

[0015] As used herein, “GG <2 prostate cancer” means “Grade Group <2 prostate cancer”. In some embodiments, the GG <2 prostate cancer is GG 1 prostate cancer. In some embodiments, the GG <2 prostate cancer is no prostate cancer.

[0016] As used herein, the term “whole urine” refers to complete, undiluted output from the subject’s urinary system. The whole urine used in the methods described herein is not further processed (e.g., fractionated by centrifugation or filtered to remove sediment or debris) from the subject’s urine sample prior to admixing the whole urine with the first solid support.

[0017] As used herein, the term “first-catch urine” (also known as “first-void urine” or “first-pass urine”) refers to the first up to about 40 mL of urine passed by a subject on the day that the subject provides a urine sample.

[0018] As used herein, the term “about” means ± 10% variation from an immediately following numerical value unless otherwise indicated. Where the term “about” is present immediately before a numerical value, the present disclosure also includes the specific numerical value itself, unless specifically stated otherwise.

[0019] As used herein, a “prostate biopsy -naive” subject is a male human subject who has a prostate and who has not had a prostate biopsy prior to providing a urine sample useful in the present methods.

[0020] As used herein, a “prostate biopsy-prior negative” subject is a male human subject who has a prostate and who has had one or more prostate biopsies, none of which was positive for GG >1 prostate cancer.I. Extraction of RNA from whole urine

[0021] The methods described herein comprise extracting RNA present in whole urine from a subject’s urine sample. In some embodiments, the whole urine comprises first-catch urine. In some embodiments, the whole urine is first-catch urine.

[0022] In some embodiments, the whole urine is decanted or otherwise removed, e.g., by pipetting, from the urine sample. In some embodiments, the whole urine has a volume of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, or about 15 mL. In some embodiments, the whole urine has a volume of about 5 mL to about 10 mL. In some embodiments, the whole urine has a volume of about 5 mL. In some embodiments, the methods comprise admixing the whole urine and the first solid support to provide the RNA-bound first solid support. In some embodiments, the first solid support is one of a plurality of first solid supports, the RNA-bound first solid support is one of a plurality of RNA-bound first solid supports, and the methods comprise admixing the whole urine and the plurality of first solid supports to provide the plurality of RNA- bound first solid supports. In some embodiments, the methods comprise admixing whole urine, a preservative and the first solid support to provide the RNA-bound first solid support. In some embodiments, the methods comprise admixing the whole urine, a preservative and a plurality of first solid supports to provide a plurality of RNA-bound first solid supports. In some embodiments, the preservative is Urine Conservation Medium (UCM). In some embodiments, the preservative is Urinary Analyte Stabilizer (UAS).

[0023] In some embodiments, the methods described herein do not comprise isolating an exosome, e.g., an exosome comprising an RNA, from the whole urine or the subject’s urine sample. Without being bound by theory, it is believed that avoiding an exosome-isolation step makes the sample processing more robust (e.g., less likely to fail) and obviates a requirement to preserve exosomes in the urine prior to RNA extraction, both streamlining sample processing and reducing processing cost per sample.

[0024] In some embodiments, admixing the whole urine and the first solid support comprises adding the whole urine to the first solid support, or vice versa, e.g., by pipetting. In some embodiments, admixing the whole urine and the plurality of first solid supports comprises adding the whole urine to the plurality of first solid supports, or vice versa, e.g., by pipetting. In some embodiments, the first solid support comprises a silicon carbide surface for interaction with RNA, which can be bound to the silicon carbide surface by, e.g., adsorption.

[0025] In some embodiments, the first solid support is silicon carbide. In some embodiments, the first solid support is a silicon carbide particle. In some embodiments, the silicon carbide particle is a silicon carbide nanoparticle. In some embodiments, the silicon carbide particle is one of a plurality of silicon carbide particles, and the methods comprise admixing the whole urine and the plurality of silicon carbide particles. In some embodiments, the silicon carbide nanoparticle is one of a plurality of silicon carbide nanoparticles, and the methods comprise admixing the whole urine and the plurality of silicon carbide particles. In some embodiments, the plurality of silicon carbide particles, e.g., silicon carbide nanoparticles, are present in a slurry, and the methods comprise admixing the whole urine and the slurry. In some embodiments, the plurality of silicon carbide particles is Slurry C3 commercially available from Norgen Biotek Corp. In some embodiments, the methods comprise admixing about 0.07 mL of slurry for every 1 mL of whole urine. In some embodiments, the methods comprise admixing about 0.35 mL of slurry for every 5 mL of whole urine. In some embodiments, the methods comprise admixing the whole urine and the slurry at a ratio of about 1 to about 0.05 (v / v) whole urine to slurry, about 1 to about 0.06 (v / v) whole urine to slurry, about 1 to about 0.07 (v / v) whole urine to slurry, about 1 to about 0.08 (v / v) whole urine to slurry, about 1 to about 0.09 (v / v) whole urine to slurry, or about 1 to about 0.1 (v / v) whole urine to slurry. In some embodiments, the admixing the whole urine and the slurry occurs at a concentration of about 1 to about 0.07 (v / v) whole urine to slurry.

[0026] In some embodiments, the first solid support is one of a plurality of silicon carbide particles, and the methods comprise admixing about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg of the plurality of silicon carbide particles per mL of whole urine. In some embodiments, the methods comprise admixing about 100 mg of the plurality of silicon carbide particles per mL of whole urine. In some embodiments, the plurality of silicon carbide particles is present in a slurry.

[0027] In some embodiments, the first solid support is contained in a column, and the methods comprise admixing within the column the whole urine and the first solid support. In some embodiments, the plurality of first solid supports is contained in a column, and the methods comprise admixing within the column the whole urine and the plurality of first solid supports. In some embodiments, the plurality of first solid supports is a plurality of silicon carbide particles, and the column contains the plurality of silicon carbide particles. In some embodiments, the plurality of silicon carbide particles is present in a slurry.

[0028] In some embodiments, the methods comprise admixing the whole urine, the first solid support and a lysis buffer. In some embodiments, the methods comprise admixing the whole urine,the plurality of first solid supports and a lysis buffer. In some embodiments, the plurality of first solid supports, e.g., silicon carbide particles, is present in a slurry.

[0029] In some embodiments, the methods comprise admixing whole urine, a preservative, a lysis buffer, and the first solid support to provide the RNA-bound first solid support. In some embodiments, the methods comprise admixing whole urine, a preservative, a lysis buffer, and a plurality of first solid supports to provide a plurality of RNA-bound first solid supports. In some embodiments, the preservative is UCM. In some embodiments, the preservative is UAS.

[0030] Lysis buffers suitable for the methods described herein include, for example, and without limitation, nonionic and ionic detergents or surfactants. Illustrative nonionic surfactants include, but are not limited to, t-octylphenoxypolyethoxyethanol (e.g., TRITON X-100),(octylphenoxy )poly ethoxy ethanol (e.g., IGEPALCA-630 / NP-40), tri ethyleneglycol monolauryl ether (e.g., BRIJ 30), sorbitan monolaurate (e.g., SPAN 20), or the polysorbate family of chemicals, such as polysorbate 20 (e.g., TWEEN 20). Other commercially available Polysorbates include TWEEN 40, TWEEN 60 and TWEEN 80 (Sigma-Aldrich, St. Louis, MO). Ionic detergents, such as sodium dodecyl sulfate (SDS) can be used in sample preparations for nucleic acid. The lysis buffer can optionally comprise a chaotropic salt, such as guanidinium thiocyanate or guanidinium chloride. In some embodiments, the lysis buffer comprises phenol. In some embodiments, the lysis buffer does not comprise phenol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P-mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium chloride. In some embodiments, the lysis buffer comprises guanidinium chloride and P-mercaptoethanol. In some embodiments, the lysis buffer is a phenol-based lysis buffer.

[0031] In some embodiments, the lysis buffer comprises a reducing agent, e.g., 2-aminoethanethiol, tris-carboxyethylphosphine (TCEP), or P-mercaptoethanol. In some embodiments, the lysis buffer comprises P-mercaptoethanol. In some embodiments, the P-mercaptoethanol is present in the lysis buffer at a concentration of up to about 5% by volume of the lysis buffer. In some embodiments, the lysis buffer comprises P-mercaptoethanol at a concentration of about 1% to about 2% by volume of the lysis buffer. In some embodiments, the P-mercaptoethanol is present in the lysis buffer at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% by volume of the lysis buffer. In some embodiments, the P- mercaptoethanol is present in the lysis buffer at a concentration of about 1% by volume of the lysis buffer. In some embodiments, the subject did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

[0032] In some embodiments, the methods comprise admixing the RNA-bound first solid support and a polar organic solvent to provide an RNA-bound first solid support admixture. In some embodiments, the RNA-bound first solid support is a plurality of RNA-bound first solid supports, and the methods comprise admixing the plurality of RNA-bound first solid supports and a polar organic solvent to provide an RNA-bound first solid support admixture. In some embodiments, the polar organic solvent is acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, ethyl acetate, ethylene glycol or a C1-C4 alcohol. In some embodiments, the polar organic solvent is a C1-C4 alcohol, and the C1-C4 alcohol is methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol or tert-butanol. In some embodiments, the polar organic solvent is a C1-C4 alcohol, the C1-C4 alcohol is ethanol and the ethanol is 190 proof ethanol, >95.0% ethanol, >99.5% ethanol, >99.8% ethanol, >99.9% ethanol or 100% ethanol. In some embodiments, the polar organic solvent is 100% ethanol. In some embodiments, the volume of the polar organic solvent, e.g., ethanol, is about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL. In some embodiments, the admixing the RNA-bound first solid support and a polar organic solvent comprises admixing, e.g., vortexing, the RNA-bound first solid support and the polar organic solvent, or admixing, e.g., vortexing, the plurality of RNA-bound first solid supports and polar organic solvent, for at least about 5 seconds, at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 25 seconds, or at least about 30 seconds. In some embodiments, the admixing the RNA-bound first solid support and a polar organic solvent comprises admixing, e.g., vortexing, the RNA-bound first solid support and the polar organic solvent, or admixing, e.g., vortexing, the plurality of RNA-bound first solid supports and polar organic solvent, for about 10 seconds.

[0033] In some embodiments, the methods comprise performing a centrifugation of the RNA-bound first solid support admixture to provide a pellet and a supernatant. In some embodiments, the RNA- bound first solid support admixture comprises an RNA-bound first solid support. In some embodiments, the RNA-bound first solid support admixture comprises a plurality of RNA-bound first solid supports.

[0034] In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture is for about 5 minutes.

[0035] In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture comprises subjecting the RNA-bound first solid support admixture to a centrifugalforce of from about 500 x g to about 5,000 x g, from about 1,500 x g to about 3,000 x g, or from about 1,500 x g to about 2,500 x g. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture comprises subjecting the RNA-bound first solid support admixture to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the RNA- bound first solid support admixture comprises subjecting the RNA-bound first solid support admixture to a centrifugal force of about 2,000 x g.

[0036] In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the RNA-bound first solid support admixture occurs at a temperature of about 30 °C.

[0037] In some embodiments, the supernatant is removed and optionally discarded. In some embodiments, the supernatant is removed by decanting. In some embodiments, the supernatant is removed by pipetting.

[0038] In some embodiments, the methods comprise removing the supernatant, and washing the pellet with a wash buffer to provide a washed RNA-bound first solid support. In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, and the methods comprise removing the supernatant, and admixing the pellet and a wash buffer to provide the plurality of washed RNA-bound first solid supports. In some embodiments, the wash buffer has a volume of about 50 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, about 500 pL, about 550 pL, about 600 pL, about 650 pL, about 700 pL, about 750 pL, about 800 pL, about 850 pL, about 900 pL, about 950 pL, about 1000 pL, or more. In some embodiments, the wash buffer has a volume of about 500 pL. In some embodiments, the washing the pellet with the wash buffer comprises pipetting or vortexing one or both of the pellet and wash buffer. In some embodiments, the methods comprise discarding the supernatant after removing it.

[0039] In some embodiments, the methods comprise admixing a wash buffer, the washed RNA- bound first solid support, and a second solid support to provide an RNA-bound second solid support,wherein the second solid support comprises a silicon-based compound. Silicon-based compounds include, but are not limited to, silicon carbide, silica, polysilicic materials, silicates, borosilicates, inorganic glasses, and silica materials such as silica particles, silica fibers, glass fibers, glass particles, glass powders, silica sand, silica gel, diatomaceous earth, glass, alkylsilica, aluminosilicate, and borosilicate and the like. The silicon-based compound can be present in a slurry, can be in a bead-based format, or can be incorporated in one or more other structures, such as silica membranes, silica impregnated or coated filters, or silica coated magnetic beads. The second solid support described herein can be porous or non-porous, permeable or impermeable, including but not limited to, and in the form of, present in or contained in a membrane, column, slurry, resin, filter paper, sheet, particle, magnetic particle, bead, magnetic bead, gel, powder, fiber, and the like. In some embodiments, the second solid support comprises a silica surface for interaction with RNA, which can be bound to the silica surface by, e.g., adsorption. In some embodiments, the washed RNA- bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, the RNA bound second solid support is a plurality of RNA-bound second solid supports, and the methods comprise admixing a wash buffer, the plurality of washed RNA-bound first solid supports, and a plurality of second solid supports to provide a plurality of RNA-bound second solid supports.

[0040] In some embodiments, the second solid support is silica. In some embodiments, the second solid support is a silica particle. In some embodiments, the silica particle is one of a plurality of silica particles. In some embodiments, the plurality of silica particles is present in a slurry. In some embodiments, the plurality of silica particles is contained in a column.

[0041] In some embodiments, the second solid support is silicon carbide. In some embodiments, the second solid support is a silicon carbide particle. In some embodiments, the silicon carbide particle is one of a plurality of silicon carbide particles. In some embodiments, the plurality of silicon carbide particles is present in a slurry. In some embodiments, the plurality of silicon carbide particles is contained in a column.

[0042] In some embodiments, the second solid support comprises a first silicon-based compound, wherein the first silicon-based compound is silica, and further comprises a second silicon-based compound, wherein the second silicon-based compound is silicon carbide.

[0043] In some embodiments, the second solid support is one of a plurality of silica particles. In some embodiments, the second solid support is one of a plurality of silicon carbide particles.

[0044] In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of silica particles, and the methods comprise admixing a wash buffer, the plurality of washed RNA-boundfirst solid supports, and the plurality of silica particles to provide a plurality of RNA-bound second solid supports.

[0045] In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of silicon carbide particles, and the methods comprise admixing a wash buffer, the plurality of washed RNA- bound first solid supports, and the plurality of silicon carbide particles to provide a plurality of RNA- bound second solid supports.

[0046] In some embodiments, the methods comprise admixing a wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of silica particles and a plurality of silicon carbide particles to provide a plurality of RNA-bound second solid supports.

[0047] In some embodiments, the methods comprise admixing a wash buffer, a washed RNA-bound first solid support, a second solid support and a third solid support to provide an RNA-bound second solid support and an RNA-bound third solid support, wherein the second solid support comprises a silicon-based compound that is silica, and wherein the third solid support comprises a silicon-based compound that is silicon carbide.

[0048] In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, the third solid support is one of a plurality of third solid supports, and the methods comprise admixing the wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of second solid supports and the plurality of third solid support to provide a plurality of RNA-bound second solid supports and a plurality of RNA-bound third solid supports, wherein each second solid support comprises a silicon-based compound that is silica, and wherein each third solid support comprises a silicon-based compound that is silicon carbide.

[0049] In some embodiments, the plurality of second solid supports and plurality of third solid supports are present in a slurry. In some embodiments, the plurality of second solid supports and plurality of third solid supports are present in a slurry, wherein each second solid support comprises a silicon-based compound that is silica, and wherein each third solid support comprises a silicon- based compound that is silicon carbide.

[0050] In some embodiments, one or both of the second solid support and third solid support are contained in a filter, e.g., a membrane filter. In some embodiments, one or both of (i) the plurality of second solid supports and (ii) the plurality of third solid supports are contained in a filter, e.g., a membrane filter. In some embodiments, the membrane of the membrane filter is a positively charged membrane. In some embodiments, the membrane comprises cellulose. In some embodiments, the membrane comprises polyethersulfone (PES). In some embodiments, the filter is acomponent of a filter plate, e.g., a 96-well filter plate. In some embodiments, the filter is a component of a column, e.g., a spin column.

[0051] In some embodiments, one or both of the second solid support and third solid support are contained in a column. In some embodiments, one or both of (i) the plurality of second solid supports and (ii) the plurality of third solid supports are contained in a column. In some embodiments, the plurality of second solid supports and plurality of third solid supports are contained in a column, wherein each second solid support comprises a silicon-based compound that is silica, and wherein each third solid support comprises a silicon-based compound that is silicon carbide. In some embodiments, the plurality of second solid supports and plurality of third solid supports are contained in a column, wherein the plurality of second solid supports is a plurality of silica particles, and wherein the plurality of third solid supports is a plurality of silicon carbide particles. Examples of columns containing a plurality of silica particles and a plurality of silicon carbide particles and useful in the present methods are disclosed in U.S. Patent Nos. 9,845,463 incorporated by reference herein, for example, at column 4, lines 51-67; columns 5-9; and column 10, lines 1-42; and 9,422,596, incorporated by reference herein, for example at column 4, lines 43- 67; columns 5-9; and column 10, lines 1-26.

[0052] In some embodiments, the column comprises a first portion and a second portion, wherein the first portion comprises the plurality of second solid supports, the second portion comprises the plurality of third solid supports, each second solid support comprises a silicon-based compound that is silica and each third solid support comprises a silicon-based compound that is silicon carbide. In some embodiments, the first portion is a first layer, and the second portion is a second layer.

[0053] In some embodiments, the third solid support comprises a silicon-based compound that is silicon carbide. In some embodiments, the silicon carbide is a plurality of silicon carbide particles and the amount of the plurality of silicon carbide particles is about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg. In some embodiments, the amount of the plurality of silicon carbide particles is about 100 mg. In some embodiments, the plurality of silicon carbide particles is contained in a column.

[0054] In some embodiments, the column contains a filter. In some embodiments, the filter comprises or is a membrane. In some embodiments, one or both of the filter and membrane contain the plurality of second solid supports or the plurality of third solid supports. In some embodiments, one or both of the filter and membrane contain the plurality of second solid supports and the plurality of third solid supports. In some embodiments, the filter or membrane has a thickness of at least 0.5mm. In some embodiments, the filter or membrane has a pore size of < about 0.5 pm, in some embodiments, < about 0.22 pm. In some embodiments, the column is a well of a filter plate.

[0055] In some embodiments, the admixing the wash buffer, the washed RNA-bound first solid support, and the second solid support comprises performing a centrifugation of the wash buffer, the washed RNA-bound first solid support, and the second solid support to provide the RNA-bound second solid support and a flowthrough. In some embodiments, the admixing the wash buffer, the washed RNA-bound first solid support, the second solid support and the third solid support comprises performing a centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and the third to provide the RNA-bound second solid support, an RNA-bound third solid support and a flowthrough. In some embodiments, the flowthrough is removed and optionally discarded following the centrifugation of the wash buffer, the washed RNA- bound first solid support, the second solid support and optionally the third solid support.

[0056] In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support comprises subjecting the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support comprises subjecting the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support comprises subjecting the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support to a centrifugal force of about 3,146 x g. In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, and the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, and plurality of the second solid supports at a centrifugal force of about 3,000 x g or about 3,146 x g. In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, the third solid support is one of a plurality of third solid supports, and the methods comprise performing a centrifugation of the wash buffer, theplurality of washed RNA-bound first solid supports, plurality of the second solid supports and the plurality of third solid supports at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0057] In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, and the second solid support and optionally the third solid support is for about 2 minutes. In some embodiments, the performing the centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of second solid supports and the plurality of third solid supports is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of second solid supports and the plurality of third solid supports is for about 2 minutes. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, the second solid support and optionally the third solid support occurs at a temperature of about 30 °C. In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, and the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, and plurality of the second solid supports at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, and plurality of the second solid supports at a temperatureof about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, and plurality of the second solid supports at a temperature of about 30 °C. In some embodiments, the washed RNA-bound first solid support is one of a plurality of washed RNA-bound first solid supports, the second solid support is one of a plurality of second solid supports, the third solid support is one of a plurality of third solid supports, and the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of the second solid supports and the plurality of third solid supports at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA- bound first solid supports, the plurality of the second solid supports and the plurality of third solid supports at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the wash buffer, the plurality of washed RNA-bound first solid supports, the plurality of the second solid supports and the plurality of third solid supports at a temperature of about 30 °C.

[0058] In some embodiments, the methods comprise washing the RNA-bound second solid support to provide a washed RNA-bound second solid support. In some embodiments, the method comprises washing the RNA-bound second solid support and RNA-bound third solid support to provide a washed RNA-bound second solid support and a washed RNA-bound third solid support. In some embodiments, the washing the RNA-bound second solid support comprises admixing the RNA- bound second solid support and a wash buffer to provide an RNA-bound second solid support and wash buffer admixture, and performing a centrifugation of the RNA-bound second solid support and wash buffer admixture to provide the washed RNA-bound second solid support and a flowthrough. In some embodiments, the washing the RNA-bound second solid support and RNA-bound third solid support comprises admixing the RNA-bound second solid support, the RNA-bound third solid support and a wash buffer to provide an RNA-bound second solid support, RNA-bound third solid support and wash buffer admixture, and performing a centrifugation of the RNA-bound second solid support, RNA-bound third solid support and wash buffer admixture to provide the washed RNA- bound second solid support, the washed RNA-bound third solid support and a flowthrough. In some embodiments, the flowthrough is discarded. In some embodiments, the wash buffer has a volume of about 50 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, about 500 pL, about 550 pL, about 600 pL, about 650 pL, about 700 pL, about 750 pL, about 800 pL, about 850 pL, about 900 pL, about 950 pL, about 1000 pL, or more. In some embodiments, the wash buffer has a volume of about 400 pL. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound secondsolid supports. In some embodiments, the methods comprise washing a plurality of RNA-bound second solid supports to provide a plurality of washed RNA-bound second solid supports. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA- bound second solid supports, and the RNA-bound third solid support is one of a plurality of RNA- bound third solid supports. In some embodiments, the methods comprise washing the plurality of RNA-bound second solid supports and the plurality of RNA-bound third solid supports to provide a plurality of washed RNA-bound second solid supports and a plurality of washed RNA-bound third solid supports.

[0059] In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support and the wash buffer admixture to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support and the wash buffer admixture to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support and the wash buffer admixture to a centrifugal force of about 3,146 x g. In some embodiments, the RNA-bound second solid support is one of a plurality of RNA-bound second solid supports, and the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports and the wash buffer admixture at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0060] In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture comprises subjecting the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture to a centrifugal force of about 3,146 x g. In some embodiments, the RNA-bound second solid support is one of a plurality of RNA-bound second solidsupports, the RNA-bound third solid support is one of a plurality of RNA-bound third solid supports, and the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports, the RNA-bound third solid supports and the wash buffer admixture at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0061] In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture occurs at a temperature of about 30 °C. In some embodiments, the RNA-bound second solid support is one of a plurality of RNA-bound second solid supports, and the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports and the wash buffer admixture at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports and the wash buffer admixture at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports and the wash buffer admixture at a temperature of about 30 °C.

[0062] In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA- bound second solid support, the RNA-bound third solid support and the wash buffer admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture occurs at a temperature of about30 °C. In some embodiments, the RNA-bound second solid support is one of a plurality of RNA- bound second solid supports, the RNA-bound third solid support is one of a plurality of RNA-bound third solid supports, and the methods comprise performing a centrifugation of the plurality of RNA- bound second solid supports, the plurality of RNA-bound third solid supports and the wash buffer admixture at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports and the wash buffer admixture at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of RNA-bound second solid supports, the plurality of RNA-bound third solid supports and the wash buffer admixture at a temperature of about 30 °C.

[0063] In some embodiments, the performing the centrifugation of the RNA-bound second solid support and the wash buffer admixture is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the RNA- bound second solid support and the wash buffer admixture is for about 2 minutes.

[0064] In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the RNA-bound second solid support, the RNA-bound third solid support and the wash buffer admixture is for about 2 minutes.

[0065] In some embodiments, the methods comprise treating the washed RNA-bound second solid support with DNase to provide a DNase-treated second solid support and a flowthrough. In some embodiments, the method further comprises treating the washed RNA-bound second solid support and washed RNA-bound third solid support with deoxyribonuclease (DNase) to provide a DNase- treated second solid support, a DNase-treated third solid support and a flowthrough. In some embodiments, the treating comprises performing a centrifugation of the washed RNA-bound second solid support and DNase to provide the DNase-treated second solid support and the flowthrough. In some embodiments, the treating comprises performing a centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and DNase to provide the DNase- treated second solid support, the DNase-treated third solid support and the flowthrough. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA- bound second solid supports, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, and the treating comprises performing a centrifugation of the plurality of washed RNA-bound second solid supports and DNase to provide the plurality of DNase-treatedsecond solid supports and the flowthrough. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound second solid supports, the washed RNA- bound third solid support is one of a plurality of washed RNA-bound third solid supports, and the treating comprises performing a centrifugation of the plurality of washed RNA-bound second solid supports, the plurality of washed RNA-bound third solid supports and DNase to provide the plurality of DNase-treated second solid supports, the plurality of DNase-treated third solid supports and the flowthrough.

[0066] In some embodiments, the DNase is DNase I. In some embodiments, the DNase is RNase- free DNase. In some embodiments, the DNase I is RNase-free DNase I. The DNase, e.g., DNase I, is commercially available, e.g., from Norgen Biotek Corp.

[0067] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase comprises subjecting the washed RNA-bound second solid support and the DNase to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or from about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase comprises subjecting the washed RNA-bound second solid support and the DNase to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase comprises subjecting the washed RNA-bound second solid support and the DNase to a centrifugal force of about 3,146 x g. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound second solid supports, and the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports and the DNase at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0068] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase comprises subjecting the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or from about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase comprises subjecting the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA- bound third solid support and the DNase comprises subjecting the washed RNA-bound second solidsupport, the washed RNA-bound third solid support and the DNase to a centrifugal force of about 3,146 x g. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound second solid supports, the washed RNA-bound third solid support is one of a plurality of washed RNA-bound third solid supports and the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports, the plurality of washed RNA-bound third solid supports and the DNase at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0069] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 30 °C. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound second solid supports, and the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports and the DNase at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports and the DNase at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports and the DNase at a temperature of about 30 °C.

[0070] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA- bound third solid support and the DNase occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase occurs at a temperature of about23 °C. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, the washed RNA-bound third solid support and the DNase occurs at a temperature of about 30 °C. In some embodiments, the washed RNA-bound second solid support is one of a plurality of washed RNA-bound second solid supports, the washed RNA-bound third solid support is one of a plurality of washed RNA-bound third solid supports, and the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports, the plurality of washed RNA-bound third solid supports and the DNase at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports, the plurality of washed RNA-bound third solid supports and the DNase at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of washed RNA-bound second solid supports, washed RNA-bound third solid supports and the DNase at a temperature of about 30 °C.

[0071] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support and the DNase is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the washed RNA- bound second solid support and the DNase is for about 2 minutes.

[0072] In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, RNA-bound third solid support and the DNase is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the washed RNA-bound second solid support, washed RNA-bound third solid support and the DNase is for about 2 minutes.

[0073] In some embodiments, the flowthrough is a first flowthrough. In some embodiments, the treating further comprises admixing the DNase-treated second solid support and the first flowthrough to provide a DNase-treated second solid support admixture, and incubating the DNase-treated second solid support admixture to provide an incubated DNase-treated solid support admixture. In some embodiments, the treating further comprises admixing the DNase-treated second solid support, the DNase-treated third solid support and the first flowthrough to provide a DNase-treated second solid support and DNase-treated third solid support admixture, and incubating the DNase-treated second solid support and DNase-treated third solid support admixture to provide an incubated DNase-treated solid support admixture.

[0074] In some embodiments, the incubating occurs at room temperature. In some embodiments, the incubating occurs at a temperature of about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22°C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C t. In some embodiments, the incubating occurs for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In some embodiments, the incubating occurs for about 15 minutes.

[0075] In some embodiments, the methods further comprise admixing the incubated DNase-treated solid support admixture and a wash buffer to provide a DNase-treated second solid support and wash buffer admixture, and performing a centrifugation of the DNase-treated second solid support and wash buffer admixture to provide the washed DNase-treated second solid support and a second flowthrough.

[0076] In some embodiments, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, and the methods comprise washing the plurality of DNase-treated second solid supports to provide a plurality of washed DNase-treated second solid supports. In some embodiments, the DNase-treated second solid support is one of a plurality of DNase-treated second solid supports, the DNase-treated third solid support is one of a plurality of DNase-treated third solid supports, and the methods comprise washing the plurality of DNase-treated second solid supports and the plurality of DNase-treated third solid supports to provide a plurality of washed DNase- treated second solid supports and a plurality of washed DNase-treated third solid supports.

[0077] In some embodiments, the methods further comprise admixing the incubated DNase-treated support admixture and a wash buffer to provide a DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture, and performing a centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture to provide the washed DNase-treated second solid support, the washed DNase-treated third solid support and a second flowthrough.

[0078] In some embodiments, the methods further comprise removing and optionally discarding one or more of the flowthrough, first flowthrough and second flowthrough after performing the centrifugation. In some embodiments, one or more of the flowthrough, first flowthrough and second flowthrough are removed by pipetting. In some embodiments, one or more of the flowthrough, first flowthrough and second flowthrough are removed by decanting. In some embodiments, the wash buffer has a volume of about 50 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, about 500 pL, about 550 pL, about 600 pL, about 650 pL, about 700 pL, about 750 pL, about 800 pL, about 850 pL, about 900 pL, about 950 pL, about 1000 pL, or more. In some embodiments, the wash buffer has a volume of about 500 pL.

[0079] In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support andwash buffer admixture to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support and wash buffer admixture to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support and wash buffer admixture to a centrifugal force of about 3,146 x g.

[0080] In some embodiments, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, and the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports and wash buffer admixture at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0081] In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture to a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture to a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture comprises subjecting the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture to a centrifugal force of about 3,146 x g.

[0082] In some embodiments, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, the DNase-treated third solid support is one of a plurality of DNase- treated third solid supports, and the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports, the plurality of DNase-treated third solid supports and wash buffer admixture at a centrifugal force of about 3,000 x g or about 3,146 x g.

[0083] In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. Insome embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 23 °C. In some embodiments, the performing a centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 30 °C.

[0084] In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the DNase- treated second solid support, DNase-treated third solid support and wash buffer admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the DNase-treated second solid support, DNase-treated third solid support and wash buffer admixture occurs at a temperature of about 23 °C. In some embodiments, the performing a centrifugation of the DNase-treated second solid support, DNase- treated third solid support and wash buffer admixture occurs at a temperature of about 30 °C.

[0085] In some embodiments, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, and the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports and wash buffer admixture at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports and wash buffer admixture at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports and wash buffer admixture at a temperature of about 30 °C.

[0086] In some embodiments, the DNase-treated second solid support is one of a plurality of DNase- treated second solid supports, the DNase-treated third solid support is one of a plurality of DNase- treated third solid supports, and the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports, plurality of DNase-treated third solid supports and wash buffer admixture at a temperature of about 23 °C to about 30 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality of DNase-treated second solid supports, plurality of DNase-treated third solid supports and wash buffer admixture at a temperature of about 23 °C. In some embodiments, the methods comprise performing a centrifugation of the plurality ofDNase-treated second solid supports, plurality of DNase-treated third solid supports and wash buffer admixture at a temperature of about 30 °C.

[0087] In some embodiments, the performing the centrifugation is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing a centrifugation of the DNase-treated second solid support and the wash buffer admixture is for about 2 minutes.

[0088] In some embodiments, the methods further comprise repeating the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support. In some embodiments, the methods comprise repeating once the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support, and, accordingly, comprise these two admixing steps. In some embodiments, the methods comprise repeating only once the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support, and, accordingly, comprise no more than these two admixing steps. In some embodiments, the methods comprise repeating twice the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support, and accordingly, comprise these three admixing steps. In some embodiments, the methods comprise repeating only twice the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support, and, accordingly, comprise no more than these three admixing steps.

[0089] In some embodiments, the methods further comprise repeating the steps of admixing the plurality of DNase-treated second solid supports and the wash buffer and performing a centrifugation to provide a plurality of washed DNase-treated second solid supports. In some embodiments, the methods comprise repeating once the steps of admixing the plurality of DNase-treated second solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports, and, accordingly, comprise these two admixing steps. In some embodiments, the methods comprise repeating only once the steps of admixing the plurality of DNase-treated second solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports, and, accordingly, comprise no more than these two admixing steps. In some embodiments, the methods comprise repeating twice the steps of admixing the plurality of DNase-treated second solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports,and accordingly, comprise these three admixing steps. In some embodiments, the methods comprise repeating only twice the steps of admixing the plurality of DNase-treated second solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports, and, accordingly, comprise no more than these three admixing steps.

[0090] In some embodiments, the methods further comprise repeating the steps of admixing the DNase-treated second solid support, the DNase-treated third solid support and the wash buffer, and performing a centrifugation to provide the washed DNase-treated second solid support and the washed DNase-treated third solid support. In some embodiments, the methods comprise repeating once the steps of admixing the DNase-treated second solid support, the DNase-treated third solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support and the washed DNase-treated third solid support, and, accordingly, comprise these two admixing steps. In some embodiments, the methods comprise repeating only once the steps of admixing the DNase-treated second solid support, the DNase-treated third solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support and the washed DNase-treated third solid support, and, accordingly, comprise no more than these two admixing steps. In some embodiments, the methods comprise repeating twice the steps of admixing the DNase-treated second solid support, the DNase-treated third solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support and the washed DNase-treated third solid, and, accordingly, comprise these three admixing steps. In some embodiments, the methods comprise repeating only twice the steps of admixing the DNase- treated second solid support, the DNase-treated third solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support and the washed DNase-treated third solid, and, accordingly, comprise no more than these three admixing steps.

[0091] In some embodiments, the methods further comprise repeating the steps of admixing the plurality of DNase-treated second solid supports, the plurality of DNase-treated third solid supports and the wash buffer, and performing a centrifugation to provide the plurality of washed DNase- treated second solid supports and the plurality of washed DNase-treated third solid supports. In some embodiments, the methods comprise repeating once the steps of admixing the plurality of DNase- treated second solid supports, the plurality of DNase-treated third solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports and the plurality of washed DNase-treated third solid supports, and, accordingly, comprise these two admixing steps. In some embodiments, the methods comprise repeating only once the steps of admixing the plurality of DNase-treated second solid supports, the plurality of DNase-treated thirdsolid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports and the plurality of washed DNase-treated third solid supports, and, accordingly, comprise no more than these two admixing steps. In some embodiments, the methods comprise repeating twice the steps of admixing the plurality of DNase-treated second solid supports, the plurality of DNase-treated third solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase-treated second solid supports and the plurality of washed DNase-treated third solids, and, accordingly, comprise these three admixing steps. In some embodiments, the methods comprise repeating only twice the steps of admixing the plurality of DNase-treated second solid supports, the plurality of DNase-treated third solid supports and the wash buffer and performing a centrifugation to provide the plurality of washed DNase- treated second solid supports and the plurality of washed DNase-treated third solid supports, and, accordingly, comprise no more than these three admixing steps.

[0092] In some embodiments, the methods comprise drying the washed DNase-treated second solid support to provide a dried second solid support. In some embodiments, the drying comprises performing a centrifugation of the washed DNase-treated solid support to provide a dried second solid support. In some embodiments, the dried second solid support comprises, e.g., on its surface, no detectable amount of remaining polar organic solvent, e.g., ethanol. In some embodiments, the dried second solid support is one of a plurality of dried second solid supports. In some embodiments, the methods comprise drying a plurality of washed DNase-treated solid supports to provide a plurality of dried second solid supports.

[0093] In some embodiments, the methods comprise drying the washed DNase-treated second solid support and the washed DNase-treated third solid support to provide a dried second solid support and a dried third solid support. In some embodiments, the drying comprises performing a centrifugation of the washed DNase-treated second solid support and the washed DNase-treated third solid support to provide the dried second solid support and the dried third solid support. In some embodiments, either the dried second solid support or the dried third solid support comprises, e.g., on its surface, a detectable amount of remaining polar organic solvent, e.g., ethanol. In some embodiments, neither the dried second solid support nor the dried third solid support comprises, e.g., on its surface, any detectable amount of remaining polar organic solvent, e.g., ethanol. In some embodiments, the dried second solid support is one of a plurality of dried second solid supports, and the dried third solid support is one of a plurality of dried third solid supports. In some embodiments, the methods comprise drying a plurality of washed DNase-treated second solid supports and a plurality of washed DNase-treated third solid supports to provide a plurality of dried second solid supports and a plurality of dried third solid supports.

[0094] In some embodiments, the drying comprises performing a centrifugation of the washed DNase-treated second solid support, and optionally the washed DNase-treated third solid support, at a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or about 2,500 x g to about 3,500 x g. In some embodiments, the drying comprises performing the centrifugation at a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the drying comprises performing the centrifugation at a centrifugal force of about 3,146 x g.

[0095] In some embodiments, the washed DNase-treated second solid support is one of a plurality of washed DNase-treated second solid supports, and the methods comprise performing a centrifugation of the plurality of washed DNase-treated second solid supports at a centrifugal force of about 3,000 x g (e.g., 3,146 x g). In some embodiments, the washed DNase-treated second solid support is one of a plurality of washed DNase-treated second solid supports, the washed DNase-treated third solid support is one of a plurality of washed DNase-treated third solid supports, and the methods comprise performing a centrifugation of the plurality of washed DNase-treated second solid supports and the plurality of DNase-treated third solid supports at a centrifugal force of about 3,000 x g e.g., 3,146 x g)-

[0096] In some embodiments, the drying comprises performing the centrifugation for up to about 30 minutes. In some embodiments, the drying comprises performing the centrifugation for up to about 20 minutes. In some embodiments, the drying comprises performing the centrifugation for about 5 minutes to about 30 minutes. In some embodiments, the drying comprises performing the centrifugation for about 10 minutes to about 20 minutes. In some embodiments, the drying comprises performing the centrifugation for about 5 minutes to about 15 minutes. In some embodiments, the drying comprises performing the centrifugation for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, or more. In some embodiments, the drying comprises performing the centrifugation for about 15 minutes.

[0097] In some embodiments, the drying comprises performing the centrifugation at a temperature of about 20 °C to about 30 °C. In some embodiments, the drying comprises performing the centrifugation at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the drying comprises performing the centrifugation at a temperature of about 23 °C toabout 30 °C. In some embodiments, the drying comprises performing the centrifugation at a temperature of about 23 °C. In some embodiments, the drying comprises performing the centrifugation at a temperature of about 30 °C.

[0098] In some embodiments, the methods comprise eluting RNA from the dried second solid support to provide extracted RNA. In some embodiments, the methods comprise eluting RNA from the dried second solid support and the dried third solid support to provide extracted RNA. In some embodiments, the eluting comprises admixing the dried second solid support and an elution buffer to provide an elution support admixture, incubating the elution support admixture to provide an incubated elution support admixture, and performing a centrifugation of the incubated elution support admixture to provide the extracted RNA. In some embodiments, the eluting comprises admixing the dried second solid support, the dried third solid support and an elution buffer to provide an elution support admixture, incubating the elution support admixture to provide an incubated elution support admixture, and performing a centrifugation of the incubated elution support admixture to provide the extracted RNA. In some embodiments, the admixing comprises allowing the elution buffer to contact the dried second solid support and optionally the dried third solid support. In some embodiments, the admixing comprises pipetting the elution buffer onto the dried second solid support and optionally onto the dried third solid support. In some embodiments, the method further comprises eluting the RNA, e.g., at least once, at least twice, at least three times, or more than three times.

[0099] In some embodiments, the dried second solid support is one of a plurality of dried second solid supports, and the eluting comprises admixing the plurality of dried second solid supports and an elution buffer to provide the elution support admixture, incubating the elution support admixture to provide the incubated elution support admixture, and performing a centrifugation of the incubated elution support admixture to provide the extracted RNA. In some embodiments, the methods comprise eluting RNA from a plurality of dried second solid supports to provide extracted RNA.

[0100] In some embodiments, the dried second solid support is one of a plurality of dried second solid supports, the dried third solid support is one of a plurality of dried third solid supports, and the eluting comprises admixing the plurality of dried second solid supports, the plurality of dried third solid supports and an elution buffer to provide the elution support admixture, incubating the elution support admixture to provide the incubated elution support admixture, and performing a centrifugation of the incubated elution support admixture to provide the extracted RNA. In some embodiments, the methods comprise eluting RNA from a plurality of dried second solid supports and a plurality of dried third solid supports to provide the extracted RNA, in some embodiments, in an elution buffer and extracted RNA admixture. In some embodiments, the extracted RNA issuspended in the elution buffer and extracted RNA admixture. In some embodiments, the extracted RNA is dissolved in the elution buffer and extracted RNA admixture.

[0101] In some embodiments, the elution buffer is an aqueous elution buffer. In some embodiments, the elution buffer is Tris-EDTA (TE) buffer. In some embodiments, the TE buffer comprises about 10 mM Tris-HCl and about 0.1 mM EDTA. In some embodiments, the TE buffer is about 10 mM Tris-HCl and about 0.1 mM EDTA. In some embodiments, the elution buffer is Tris-HCl buffer. In some embodiments, the Tris-HCl buffer comprises about lOmM Tris-HCl. In some embodiments, the elution buffer is Tris-Cl buffer. In some embodiments, the elution buffer is water. In some embodiments, the elution buffer is RNase-free water. Elution buffers are commercially available, e.g., from Thermo Fisher Scientific or Norgen.

[0102] In some embodiments, the elution buffer has a volume of about 25 pL, 50 pL, 75 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, or about 500 pL. In some embodiments, the elution buffer has a volume of about 100 pL.

[0103] In some embodiments, the elution buffer has a temperature of from about 4 °C to about 70 °C. In some embodiments, the elution buffer has a temperature of about 5° C, about 10° C, about 15° C, about 20° C, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 70° C. In some embodiments, the elution buffer has a temperature of from about 15 °C to about 37 °C. In some embodiments, the elution buffer has a temperature of from about 20 °C to about 37 °C. In some embodiments, the elution buffer has a temperature of about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, or about 37 °C. In some embodiments, the elution buffer has a temperature of about 23 °C. In some embodiments, the elution buffer has a temperature of about 30 °C. In some embodiments, the elution buffer is at room temperature.

[0104] In some embodiments, the elution buffer has a pH of from about 6.5 to about 8.5. In some embodiments, the elution buffer has a pH of from about 7 to about 8.5. In some embodiments, the elution buffer has a pH of from about 7 to about 8. In some embodiments, the elution buffer has a pH of from about 7 to about 8. In some embodiments, the elution buffer has a pH of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2 about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2 about 8.3, about 8.4, or about 8.5. In some embodiments, the elution buffer has a pH of about 7. In some embodiments, the elution buffer is water.

[0105] In some embodiments, incubating the elution support admixture is for about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, incubating the elution support admixture is for about 2 minutes.

[0106] In some embodiments, the performing the centrifugation of the incubated elution support admixture is at a centrifugal force of about 100 rpm to about 2000 rpm. In some embodiments, the performing the centrifugation of the incubated elution support admixture is at a centrifugal force of about 250 rpm to about 750 rpm. In some embodiments, the performing the centrifugation of the incubated elution support admixture is at a centrifugal force of about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, or about 1000 rpm. In some embodiments, the performing the centrifugation of the incubated elution support admixture is at a centrifugal force of about 500 rpm.

[0107] In some embodiments, the performing the centrifugation of the incubated elution support admixture is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the centrifugation of the incubated elution support admixture is for about 2 minutes.

[0108] In some embodiments, the performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C. In some embodiments, the performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 30 °C.

[0109] In some embodiments, the performing the centrifugation of the incubated elution support admixture is a first centrifugation of the incubated elution support admixture, and the method further comprises performing a second centrifugation of the incubated elution support admixture. In some embodiments, the first centrifugation is at a low speed (e.g., about 500 rpm). In some embodiments, the second centrifugation is at a high centrifugal force (e.g., about 3,000 x g).

[0110] In some embodiments, the performing the second centrifugation of the incubated elution support admixture is at a centrifugal force of from about 500 x g to about 5,000 x g, from about 2,000 x g to about 4,000 x g, or from about 2,500 x g to about 3,500 x g. In some embodiments, theperforming the second centrifugation of the incubated elution support admixture is at a centrifugal force of about 1,000 x g, about 2,000 x g, about 3,000 x g, 4,000 x g, about 5,000 x g, or more. In some embodiments, the performing the second centrifugation of the incubated elution support admixture is at a centrifugal force of about 3,146 x g.[OHl] In some embodiments, the performing the second centrifugation of the incubated elution support admixture is for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or more. In some embodiments, the performing the second centrifugation of the incubated elution support admixture is for about 2 minutes.

[0112] In some embodiments, the performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 20 °C to about 30 °C. In some embodiments, the performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In some embodiments, the performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C to about 30 °C. In some embodiments, the performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C. In some embodiments, the performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 30 °C.

[0113] Without wishing to be bound by theory, it is believed that the washing steps useful in the methods described herein can remove one or more contaminates, e.g., primarily alcohol, guanidinium chloride or guanidinium thiocyanate.

[0114] In some embodiments, the wash buffer comprises a salt. In some embodiments, the wash buffer comprises the salt at a concentration of from about 0.01 to about 2.5 M. In some embodiments, the salt is NaCl. In some embodiments, the wash buffer comprises a salt, e.g., NaCl, andtris(hydroxymethyl)aminomethane (Tris).

[0115] Suitable wash buffers can include a variety of one or more components. In some embodiments, the wash buffer comprises one or more of the following reagents: Tris, Bis-Tris, Bis- Tris-Propane, imidazole, citrate, methylmalonic acid, acetic acid, ethanol, ethanolamine, diethanolamine, triethanolamine (TEA) and sodium phosphate. In some embodiments, the wash buffer comprises ethanol.

[0116] Additional components that may be present in wash buffers include non-ionic surfactants or detergents, ionic or zwitter-ionic surfactants or detergents, chaotropic salts (e.g., guanidinium thiocyanate or guanidinium chloride), disulfide bond reducing agents, proteases, nucleases, and otheradditives or components that digest, denature, disrupt, or degrade for the purpose of extracting, purifying, enriching, or otherwise isolating nucleic acids. Non-ionic surfactants or detergents can include, but are not limited to, surfactants from the following classes: octylphenol ethoxylate, polysorbate, poloxamer, or polyoxyethylene.

[0117] Octylphenol ethoxylate surfactants can include, but are not limited to, branched octylphenoxy polyethoxy ethanol (e.g., IGEPAL CA-630), t-octylphenoxypolyethoxyethanol (e.g., Triton X-100), or other polyethylene oxide chains with an aromatic hydrocarbon lipophilic or hydrophobic group. Polysorbate surfactants can include but are not limited to polyethylene glycol sorbitan monolaurate (e.g., Tween 20), polyethylene glycol sorbitan monooleate (e.g., Tween 80), or sorbitan monooleate (e.g., Span 80). Poloxamer surfactants can include, but are not limited to, polyoxyethylenepolyoxypropylene block copolymer (e.g., Pluronic F-68) or polyethylene-polypropylene glycol block copolymer (e.g., Pluronic F-127). Polyoxyethylene surfactants can include, but are not limited to, nonylphenoxy-polyethoxylethanol (e.g., NP-40).

[0118] Non-ionic surfactants or detergents can include but are not limited to IGEPAL (e.g., IGEPAL CA-630), Triton X-100, Tween 20, Tween 80, NP-40, other block copolymers including Pluronic (e.g., F-68 or F-127), Span 80, and pegylated polymers or copolymers. Non-ionic surfactants or detergents can be used to reduce or prevent biological molecule adsorption to channel walls, or to control wetting and / or surface tension properties of fluids to control loading of sample into fluidic devices. Non-ionic surfactants or detergents can be present at concentrations from about 0.0005-5 % v / v or w / v. Ionic surfactants or detergents can include but are not limited to sodium dodecyl sulfate (e.g., at 0.01-2% w / v), sodium dodecylbenzenesulfonate (e.g., at 0.01-2% w / v), sodium cholesteryl sulfate (e.g., at 0.01%-2% w / v), and sodium deoxycholate (e.g., at about 10- 1000 mM). Chaotropic agents can include but are not limited to urea (e.g., at about 0.5-9.5 M, or in some cases, 5-9.5 M) thiourea, butanol, ethanol, guanidine, guanidinium chloride, guanidinium thiocyanate, lithium perchlorate, lithium acetate, lithium chloride, magnesium chloride, phenol, and propanol. Disulfide bond reducing agents can include, but are not limited to, DTT (e.g., at about 0.1- 40 mM, or about 10 mM) and beta-mercaptoethanol (e.g., at about 0 to about 5%). Proteases can include but are not limited to Proteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin, and papain. Nucleases can include but are not limited to non-specific nucleic acid digestion enzymes such as DNases including DNase I (e.g., to prepare DNA-free RNA extractions) and RNase, such as RNase A, RNase T, or combinations thereof (e.g., to prepare RNA- free DNA extractions). Nucleases can also include specific nucleic acid digestion enzymes (e.g., restriction enzymes) which can cut at specific nucleic acid sequences and can produce predictable fragment sizes and fragment size distributions.

[0119] In some embodiments, where the extracted RNA is present in an elution buffer and extracted RNA admixture, the extracted RNA concentration can is increased, for example, comprising precipitating the extracted RNA to provided precipitated RNA and subsequently reconstituting the precipitated RNA using a relatively smaller volume of buffer. In some embodiments, the extracted RNA concentration can be increased, for example, by using a vacuum concentrator.

[0120] In some embodiments, the extracted RNA is not separated by size prior to its detection. In some embodiments, the extracted RNA is not concentrated prior to its detection.

[0121] In some embodiments, the extracted RNA is provided in an amount that is at least 1.5-fold greater, 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6- fold greater, or more, than extracted RNA obtained from a method that does not comprise admixing whole urine and a solid support comprising silicon carbide.

[0122] Also provided are compositions comprising whole urine and a solid support comprising silicon carbide. In some embodiments, the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample. In some embodiments, the solid support is one of a plurality of solid supports, and the composition comprises whole urine and the plurality of solid supports. In some embodiments, the plurality of solid supports is a plurality of silicon carbide particles. In some embodiments, the compositions further comprise a preservative. In some embodiments, the preservative is UCM. In some embodiments, the preservative is UAS. In some embodiments, the compositions further comprise a lysis buffer. In some embodiments, the lysis buffer comprises guanidinium thiocyanate, guanidinium chloride or P-mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P-mercaptoethanol.

[0123] Further provided are compositions comprising urine and an RNA-bound solid support. In some embodiments, the RNA-bound support is an mRNA-bound support. In some embodiments, the RNA-bound support is one of a plurality of RNA-bound solid supports, and the composition comprises the plurality of RNA-bound supports. In some embodiments, the compositions further comprise a preservative. In some embodiments, the preservative is UCM. In some embodiments, the preservative is UAS. In some embodiments, the compositions further comprise a lysis buffer. In some embodiments, the lysis buffer comprises guanidinium thiocyanate, guanidinium chloride or P- mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P- mercaptoethanol.

[0124] In some embodiments, the compositions are made by a method comprising admixing whole urine and a solid support comprising silicon carbide to provide the RNA-bound solid support,wherein the admixing is not performed under reduced pressure. In some embodiments, the compositions are made by a method comprising admixing whole urine and a plurality of solid supports comprising silicon carbide to provide the plurality of RNA-bound solid supports, wherein the admixing is not performed under reduced pressure. In some embodiments, the reduced pressure is about 0.8 bar. In some embodiments, the method comprises admixing whole urine, a preservative, a lysis buffer, and a plurality of solid supports comprising silicon carbide, wherein the admixing is not performed under reduced pressure. In some embodiments, the method comprises admixing whole urine, a preservative, a lysis buffer, and silicon carbide particles, wherein the admixing is not performed under reduced pressure. In some embodiments, the lysis buffer comprises guanidinium thiocyanate, guanidinium chloride or P-mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P-mercaptoethanol. In some embodiments, the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

[0125] In some embodiments, the present invention provides methods for making an RNA-bound solid support, comprising admixing whole urine and a solid support comprising silicon carbide to provide the RNA-bound solid support. In some embodiments, the methods comprise admixing whole urine, a preservative, a lysis buffer, and a solid support comprising silicon carbide. In some embodiments, the solid support is a silicon carbide particle. In some embodiments, the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample. In some embodiments, the method does not comprise admixing the whole urine and the solid support under reduced pressure. In some embodiments, the reduced pressure is about 0.8 bar. In some embodiments, the lysis buffer comprises guanidinium thiocyanate, guanidinium chloride or P- mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P- mercaptoethanol.

[0126] In some embodiments, the present invention provides methods for making a plurality of RNA-bound solid supports, comprising admixing whole urine and a plurality of solid supports comprising silicon carbide to provide the plurality of RNA-bound solid supports. In some embodiments, the methods comprise admixing whole urine, a preservative, a lysis buffer, and a plurality of solid supports comprising silicon carbide. In some embodiments, the methods comprise admixing whole urine, a preservative, a lysis buffer, and a plurality of solid supports comprising silicon carbide. In some embodiments, the plurality of solid supports is a plurality of silicon carbide particles. In some embodiments, the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180minutes before providing the urine sample. In some embodiments, the method does not comprise admixing whole urine and the plurality of solid supports under reduced pressure. In some embodiments, the reduced pressure is about 0.8 bar. In some embodiments, the lysis buffer comprises guanidinium thiocyanate, guanidinium chloride or P-mercaptoethanol. In some embodiments, the lysis buffer comprises guanidinium thiocyanate and P-mercaptoethanol.

[0127] Further provided herein are methods for detecting in urine from a urine sample provided by a subject RNA encoded by a gene of the subject, the methods comprising: allowing about 5 mL to about 10 mL of the urine from the urine sample to contact a silicon- based solid support to provide a urine admixture comprising an RNA-bound silicon-based solid support; isolating the RNA-bound silicon-based solid support from the urine admixture to provide an isolated RNA-bound silicone-based solid support; isolating the RNA from the isolated RNA-bound silicon-based solid support to provide isolated RNA; and detecting the isolated RNA, wherein the subject has not had a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.II. Detecting extracted RNA

[0128] In some embodiments, the methods described herein further comprise detecting the extracted RNA. In some embodiments, the extracted RNA comprises or is miRNA (or pre-miRNA), siRNA, circular RNA, long non-coding RNA (IncRNA), piRNA, mRNA, rRNA, tRNA, hnRNA, or noncoding RNA (ncRNA). In some embodiments, the extracted RNA is mRNA. In some embodiments, the extracted RNA is miRNA. In some embodiments, the extracted RNA is siRNA. In some embodiments, the extracted RNA is circular RNA. In some embodiments, the extracted RNA is IncRNA. In some embodiments, the extracted RNA is piRNA. In some embodiments, the extracted RNA is rRNA. In some embodiments, the extracted RNA is tRNA. In some embodiments, the extracted RNA is hnRNA. In some embodiments, the extracted RNA is ncRNA.Detecting Gene Expression

[0129] In some embodiments, detecting the extracted RNA comprises detecting an amount of expression of the subject’s gene. In some embodiments, detecting the extracted RNA comprises detecting an amount of expression of RNA transcribed from the subject’s gene. In some embodiments, detecting the extracted RNA comprises detecting the amount of mRNA transcribedfrom the gene. In some embodiments, detecting the extracted RNA comprises reverse transcribing the extracted RNA to complementary DNA (cDNA) using reverse transcriptase and detecting the cDNA. In some embodiments, detecting the extracted RNA comprises reverse transcribing the extracted RNA to cDNA using reverse transcriptase and detecting the cDNA using quantitative PCR (qPCR).

[0130] In some embodiments, the gene is a cancer gene, e.g., a prostate cancer gene, a bladder cancer gene, or a kidney cancer gene.

[0131] In some embodiments, the gene is a gene of a pathogen, e.g., a sexually transmitted infection (STI) pathogen or a urinary tract infection (UTI) pathogen. In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is of the genus Escherichia, Klebsiella, Proteus, Enterococcus, Staphylococcus, Pseudomonas, Staphylococcus, Streptococcus, or Staphylococcus. In some embodiments, the bacterium is Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Streptococcus agalactiae or Staphylococcus aureus. In some embodiments, the pathogen is a yeast. In some embodiments, the yeast is of the genus Candida. In some embodiments, the yeast is Candida albicans. In some embodiments, the gene is a gene of a pathogen and the gene is 16S rRNA. In some embodiments, the gene is of a pathogen and the gene is 23S rRNA. In some embodiments, the gene is of a pathogen and the gene is 26S rRNA. In some embodiments, the gene is of the subject’s immune response to the pathogen, e.g., a gene of the subject’s adaptive (B cell or T cell) or innate (neutrophil) immune response to the pathogen.

[0132] In some embodiments, the gene is a prostate cancer gene.

[0133] In some embodiments, the prostate cancer gene is KLK3.

[0134] In some embodiments, the prostate cancer gene is one or more of ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRRN3A, LINC00993, RRRN1, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PLA2G7, PRCAT47, SPINK1, TDO2, TK1, IMEFF2, TRGV9.1, VSIM2R, IMPRSS2-ERG, SCHRAP1, OR51E2, APOCR PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, IMSB15A, ERG, KRK4, H0XC6, and KRK3.

[0135] In some embodiments, the prostate cancer gene is one or more of ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRIN3A, LINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PRA2G7, PRCAT47, SPINK1, TDO2, TK1, TMEFF2, TRGV9.1, and VSTM2R.

[0136] In some embodiments, the prostate cancer gene is one or more of TMPRSS2-ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, HOXC6, and KLK3.

[0137] In some embodiments, the methods described herein comprise detecting an amount of expression of a TMPRSS2-ERG gene. In some embodiments, the TMPRSS2-ERG fusion comprises exon 1 of TMPRSS2 and exons 4-11 of ERG. In some embodiments, the TMPRSS2-ERG gene fusion comprises a fusion of the nucleotide sequences of Ensembl gene identifiers ENSG00000184012 and ENSG00000157554. In some embodiments, the TMPRSS2-ERG gene fusion comprises the nucleotide sequence of SEQ ID NO: 1 or a variant thereof.

[0138] In some embodiments, the methods described herein comprise detecting an amount of expression of a SCHLAP1 gene. In some embodiments, the SCHLAP1 gene comprises the nucleotide sequence provided by the HUGO Gene Nomenclature Committee (HGNC). In some embodiments, the HGNC identifier for SCHLAP1 is 48603. In some embodiments, the SCHLAP1 gene is located at chromosome position 2q31.3. In some embodiments, a SCHLAP1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000281131. In some embodiments, SCEILAPl gene comprises the nucleotide sequence of SEQ ID NO: 2 or a variant thereof.

[0139] In some embodiments, the methods described herein comprise detecting an amount of expression of a OR51E2 gene. In some embodiments, the OR51E2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for OR51E2 is 15195. In some embodiments, the OR51E2 gene is located at chromosome position 1 Ip 15.4. In some embodiments, an OR51E2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000167332. In some embodiments, an OR51E2 gene comprises the nucleotide sequence of SEQ ID NO: 3 or a variant thereof.

[0140] In some embodiments, the methods described herein comprise detecting an amount of expression of an APOCI gene. In some embodiments, the APOCI gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for APOCI is 607. In some embodiments, the APOCI gene is located at chromosome position 19ql3.32. In some embodiments, an APOCI gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000130208. In some embodiments, an APOCI gene comprises the nucleotide sequence of SEQ ID NON or a variant thereof.

[0141] In some embodiments, the methods described herein comprise detecting an amount of expression of aPCAT14 gene. In some embodiments, the PCAT14 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PCAT14 is 48977. In some embodiments, the PCAT14 gene is located at chromosome position 22ql 1.23. In someembodiments, PCAT14 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000280623. In some embodiments, PCAT14 gene comprises the nucleotide sequence of SEQ ID NO: 5 or a variant thereof.

[0142] In some embodiments, the methods described herein comprise detecting an amount of expression of a CAMKK2 gene. In some embodiments, the CAMKK2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for CAMKK2 is 1470. In some embodiments, the CAMKK2 gene is located at chromosome position 12q24.31. In some embodiments, a CAMKK2 gene comprises the nucleotide sequence of Ensembl gene ENSG00000110931. In some embodiments, a CAMKK2 gene comprises the nucleotide sequence of SEQ ID NO: 6 or a variant thereof.

[0143] In some embodiments, the methods described herein comprise detecting an amount of expression of aPCA3 gene. In some embodiments, the PCA3 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PCA3 is 8637. In some embodiments, the PCA3 gene is located at chromosome position 9q21.2. In some embodiments, PCA3 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000225937. In some embodiments, PCA3 gene comprises the nucleotide sequence of SEQ ID NO:7 or a variant thereof. In some embodiments, the method comprises detecting an amount of PCA3 at its 3' end, wherein the 3' end of PCA3 is referred to herein as “3' G43” or “PCA3.1 ” For example, and without limitation, the method can comprise detecting 3' PCA3 at one or both of its exon groups 2-3 and 3-4. In some embodiments, the methods comprise detecting an amount of expression of PCA3 at its 5' end, wherein the 5' end of PCA3 is referred to herein as “5' PCA3” or “ G43.” For example, and without limitation, the methods can comprise detecting 5' PCA3 at its exon group 1-2.

[0144] In some embodiments, the methods described herein comprise detecting an amount of expression of an NKAINl gene. In some embodiments, the NKAIN1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for NKAIN1 is 25743. In some embodiments, the NKAIN1 gene is located at chromosome position lp35.2. In some embodiments, an N KAIN 1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000084628. In some embodiments, an NKAIN1 gene comprises the nucleotide sequence of SEQ ID NO: 8 or a variant thereof.

[0145] In some embodiments, the methods described herein comprise detecting an amount of expression of B3GNT6 gene. In some embodiments, the B3GNT6 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for B3GNT6 is 24141. In some embodiments, the B3GNT6 gene is located at chromosome position 11 ql 3.5. In someembodiments, B3GNT6 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000198488. In some embodiments, B3GNT6 gene comprises the nucleotide sequence of SEQ ID NO: 9 or a variant thereof.

[0146] In some embodiments, the methods described herein comprise detecting an amount of expression of a TFF3 gene. In some embodiments, the TFF3 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TFF3 is 11757. In some embodiments, the TFF3 gene is located at chromosome position 21q22.3. In some embodiments, a TFF3 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000160180. In some embodiments, a TFF3 gene comprises the nucleotide sequence of SEQ ID NO: 10 or a variant thereof.

[0147] In some embodiments, the methods described herein comprise detecting an amount of expression of a SPON2 gene. In some embodiments, the SPON2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for SPON2 is 11253. In some embodiments, the SPON2 gene is located at chromosome position 4pl6.3. In some embodiments, a SPON2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000159674. In some embodiments, a SPON2 gene comprises the nucleotide sequence of SEQ ID NO: 11 or a variant thereof.

[0148] In some embodiments, the methods described herein comprise detecting an amount of expression of PCGEMl gene. In some embodiments, the PCGEM1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PCGEM1 is 30145. In some embodiments, the PCGEM1 gene is located at chromosome position 2q32.3. In some embodiments, PCGEMl gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000227418. In some embodiments, PCGEMl gene comprises the nucleotide sequence of SEQ ID NO: 12 or a variant thereof.

[0149] In some embodiments, the methods described herein comprise detecting an amount of expression of a TRGV9 gene. In some embodiments, the TRGV9 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TRGV9 is 12295. In some embodiments, the TRGV9 gene is located at chromosome position 7pl4.1. In some embodiments, a TRGV9 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000211695. In some embodiments, a TRGV9 gene comprises the nucleotide sequence of SEQ ID NO: 13 or a variant thereof. In some embodiments, the methods comprise detecting an amount of expression of TRGV9 at its 3' end, wherein the 3' end of TRGV9 is referred to herein as “3' TRGV9 ” For example, and without limitation, the methods can comprise detecting 3' TRGV9 at one or more of its exon groups 3-4, 4-5, and 5-6. In some embodiments, the methods comprise detectingan amount of expression of TRGV9 at its 5' end, wherein the 5' end of TRGV9 is referred to herein as “5' TRGV9 ” For example, and without limitation, the methods can comprise detecting 5' TRGV9 at its exon group 1-2.

[0150] In some embodiments, the methods described herein comprise detecting an amount of expression of a TMSB15A gene. In some embodiments, the TMSB15A gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TMSB15A is 30744. In some embodiments, the TMSB15A gene is located at chromosome position Xq22.1. In some embodiments, a TMSB15A gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000158164. In some embodiments, a TMSB15A gene comprises the nucleotide sequence of SEQ ID NO: 14 or a variant thereof.

[0151] In some embodiments, the methods described herein comprise detecting an amount of expression of an ERG gene. In some embodiments, the ERG gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for ERG is 3446. In some embodiments, the £7?G gene is located at chromosome position 21q22.2. In some embodiments, an ERG gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000157554. In some embodiments, an ERG gene comprises the nucleotide sequence of SEQ ID NO: 15 or a variant thereof.

[0152] In some embodiments, the methods described herein comprise detecting an amount of expression of & KI.K4 gene. In some embodiments, the KLK4 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for KLK4 is 6365. In some embodiments, the KLK4 gene is located at chromosome position 19ql 3.41. In some embodiments, & KI.K4 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000167749. In some embodiments, a. KLK4 gene comprises the nucleotide sequence of SEQ ID NO: 16 or a variant thereof.

[0153] In some embodiments, the methods described herein comprise detecting an amount of expression of a. HOXC6 gene. In some embodiments, the HOXC6 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for HOXC6 is 5128. In some embodiments, the HOXC6 gene is located at chromosome position 12ql3.13. In some embodiments, a. HOXC6 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000197757. In some embodiments, a. HOXC6 gene comprises the nucleotide sequence of SEQ ID NO: 17 or a variant thereof.

[0154] In some embodiments, the methods described herein comprise detecting an amount of expression of KLK3 gene. KLK3 may also be known as PSA or Prostate-Specific Antigen. In some embodiments, the KLK3 gene comprises the nucleotide sequence provided by HGNC. In someembodiments, the HGNC identifier for KLK3 is 6364. In some embodiments, the KLK3 gene is located at chromosome position 19ql3.33. In some embodiments, a KLK3 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000142515. In some embodiments, a KLK3 gene comprises the nucleotide sequence of SEQ ID NO: 18 or a variant thereof.

[0155] In some embodiments, the methods described herein comprise detecting an amount of expression of anACSMl gene. In some embodiments, the ACSMl gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for ACSM1 is 18049. In some embodiments, AXQ ACS I gene is located at chromosome position 16pl2.3. In some embodiments, anACSMl gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000166743. In some embodiments, an ACSMl gene comprises the nucleotide sequence of SEQ ID NO: 19 or a variant thereof.

[0156] In some embodiments, the methods described herein comprise detecting an amount of expression of an AMACR gene. In some embodiments, xe. AMACR gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for AMACR is 451. In some embodiments, the AMACR gene is located at chromosome position 5pl3.2. In some embodiments, an AMACR gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000242110. In some embodiments, an AMACR gene comprises the nucleotide sequence of SEQ ID NO:20 or a variant thereof.

[0157] In some embodiments, the methods described herein comprise detecting an amount of expression of an AR gene. In some embodiments, the AR gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for AR is 644. In some embodiments, the AR gene is located at chromosome position Xql2. In some embodiments, an AR gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000169083. In some embodiments, an AR gene comprises the nucleotide sequence of SEQ ID NO:21 or a variant thereof.

[0158] In some embodiments, the methods described herein comprise detecting an amount of expression of a COL9A2 gene. In some embodiments, the COL9A2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for COL9A2 is 2218. In some embodiments, the COL9A2 gene is located at chromosome position lp34.2. In some embodiments, a COL9A2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000049089. In some embodiments, an COL9A2 gene comprises the nucleotide sequence of SEQ ID NO: 22 or a variant thereof.

[0159] In some embodiments, the methods described herein comprise detecting an amount of expression of a CRISP3 gene. In some embodiments, the CRISP3 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for CRISP 3 is 16904. Insome embodiments, the CRISP3 gene is located at chromosome position 6pl2.3. In some embodiments, a CRISP 3 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000096006. In some embodiments, a CRISP3 gene comprises the nucleotide sequence of SEQ ID NO:23 or a variant thereof.

[0160] In some embodiments, the methods described herein comprise detecting an amount of expression of a CST2 gene. In some embodiments, the CST2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for CST2 is 2474. In some embodiments, the CST2 gene is located at chromosome position 20pl 1.21. In some embodiments, a CST2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000170369. In some embodiments, a CST2 gene comprises the nucleotide sequence of SEQ ID NO:24 or a variant thereof.

[0161] In some embodiments, the methods described herein comprise detecting an amount of expression of a DLX1 gene. In some embodiments, the DLX1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for DLX1 is 2914. In some embodiments, the ZI / . 7 gene is located at chromosome position 2q31.1. In some embodiments, DLX1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000144355. In some embodiments, DLX1 gene comprises the nucleotide sequence of SEQ ID NO: 25 or a variant thereof.

[0162] In some embodiments, the methods described herein comprise detecting an amount of expression of an E TV 1 gene. In some embodiments, the Z< 7'17 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for ETV1 is 3490. In some embodiments, the ETV1 gene is located at chromosome position 7p21.2. In some embodiments, an E TV 1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000006468. In some embodiments, an ETV I gene comprises the nucleotide sequence of SEQ ID NO:26 or a variant thereof.

[0163] In some embodiments, the methods described herein comprise detecting an amount of expression of an F5 gene. In some embodiments, the F5 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for F5 is 3542. In some embodiments, the F5 gene is located at chromosome position lq24.2. In some embodiments, an F5 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000198734. In some embodiments, an F5 gene comprises the nucleotide sequence of SEQ ID NO:27 or a variant thereof.

[0164] In some embodiments, the methods described herein comprise detecting an amount of expression of a GDF15 gene. In some embodiments, the GDF15 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for GDF15 is 30142. Insome embodiments, the GDF15 gene is located at chromosome position 19pl3.11. In some embodiments, a GDF15 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000130513. In some embodiments, a GDF15 gene comprises the nucleotide sequence of SEQ ID NO: 28 or a variant thereof.

[0165] In some embodiments, the methods described herein comprise detecting an amount of expression of a GLYATL1 gene. In some embodiments, the GLYATL1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for GLYATL1 is 17257. In some embodiments, the GLYATL1 gene is located at chromosome position 1 lql2.1. In some embodiments, a GLYATL1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000166840. In some embodiments, a GLYATL1 gene comprises the nucleotide sequence of SEQ ID NO:29 or a variant thereof.

[0166] In some embodiments, the methods described herein comprise detecting an amount of expression of a G0LM1 gene. In some embodiments, the G0LM1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for SPDEF is 15451. In some embodiments, the G0LM1 gene is located at chromosome position 9q21.33. In some embodiments, a G0LM1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000135052. In some embodiments, a G0LM1 gene comprises the nucleotide sequence of SEQ ID NO:30 or a variant thereof.

[0167] In some embodiments, the methods described herein comprise detecting an amount of expression of a GRIN3A gene. In some embodiments, the GRIN3A gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for GRIN3A is 16767. In some embodiments, the GRIN3A gene is located at chromosome position 9q31.1. In some embodiments, a GRIN3A gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000198785. In some embodiments, an GRIN3A gene comprises the nucleotide sequence of SEQ ID NO: 31 or a variant thereof.

[0168] In some embodiments, the methods described herein comprise detecting an amount of expression of a LINC00993 gene. In some embodiments, the LINC00993 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for LINC00993 is 48948. In some embodiments, the LINC00993 gene is located at chromosome position lOpl 1.21. In some embodiments, LINC00993 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000235687. In some embodiments, LINC00993 gene comprises the nucleotide sequence of SEQ ID NO: 32 or a variant thereof.

[0169] In some embodiments, the methods described herein comprise detecting an amount of expression of aLRRNl gene. In some embodiments, the LRRN1 gene comprises the nucleotidesequence provided by HGNC. In some embodiments, the HGNC identifier for LRRNI is 20980. In some embodiments, the LRRNI gene is located at chromosome position 3p26.2. In some embodiments, a LRRNI gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000175928. In some embodiments, an LRRNI gene comprises the nucleotide sequence of SEQ ID NO:33 or a variant thereof.

[0170] In some embodiments, the methods described herein comprise detecting an amount of expression of a MIPEP gene. In some embodiments, Ax MIPEP gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for MIPEP is 7104. In some embodiments, the MIPEP gene is located at chromosome position 13ql2.12. In some embodiments, a MIPEP gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000027003. In some embodiments, a MIPEP gene comprises the nucleotide sequence of SEQ ID NO: 34 or a variant thereof.

[0171] In some embodiments, the methods described herein comprise detecting an amount of expression of an MS4A8 gene. In some embodiments, the MS4A8 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for MS4A8 is 13380. In some embodiments, the MS4A8 gene is located at chromosome position 1 lql2.2. In some embodiments, an MS4A8 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000166959. In some embodiments, an MS4A8 gene comprises the nucleotide sequence of SEQ ID NO:35 or a variant thereof.

[0172] In some embodiments, the methods described herein comprise detecting an amount of expression of aMYO6 gene. In some embodiments, the MY06 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for MY06 is 7605. In some embodiments, the MY06 gene is located at chromosome position 6ql4.1. In some embodiments, aMYO6 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000196586. In some embodiments, aMYO6 gene comprises the nucleotide sequence of SEQ ID NO:36 or a variant thereof.

[0173] In some embodiments, the methods described herein comprise detecting an amount of expression of a CYB561A3 gene. In some embodiments, the CYB561A3 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for CYB561A3 is 23014. In some embodiments, the CYB561A3 gene is located at chromosome position 1 lql2.2. In some embodiments, a CYB561A3 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000162144. In some embodiments, a CYB561A3 gene comprises the nucleotide sequence of SEQ ID NO:37 or a variant thereof.

[0174] In some embodiments, the methods described herein comprise detecting an amount of expression of PDLIM5 gene. In some embodiments, the PDLIM5 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PDLIM5 is 17468. In some embodiments, the PDLIM5 gene is located at chromosome position 4q22.3. In some embodiments, PDIJM5 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000163110. In some embodiments, a. PDI.IM5 gene comprises the nucleotide sequence of SEQ ID NO:38 or a variant thereof.

[0175] In some embodiments, the methods described herein comprise detecting an amount of expression of a. P XK) gene. In some embodiments, the PEX10 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PEX10 is 8851. In some embodiments, the PEX10 gene is located at chromosome position lp36.32. In some embodiments, a P XlO gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000157911. In some embodiments, a P XlO gene comprises the nucleotide sequence of SEQ ID NO:39 or a variant thereof.

[0176] In some embodiments, the methods described herein comprise detecting an amount of expression of PLA1A gene. In some embodiments, the PLA1A gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PLA1A is 17661. In some embodiments, the PLA1A gene is located at chromosome position 3 ql 3.33. In some embodiments, PLA1A gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000144837. In some embodiments, PLA1A gene comprises the nucleotide sequence of SEQ ID NO:40 or a variant thereof.

[0177] In some embodiments, the methods described herein comprise detecting an amount of expression of PLA2G7 gene. In some embodiments, the PLA2G7 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PLA2G7 is 9040. In some embodiments, the PLA2G7 gene is located at chromosome position 6pl2.3. In some embodiments, PLA2G7 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000146070. In some embodiments, PLA2G7 gene comprises the nucleotide sequence of SEQ ID NON 1 or a variant thereof.

[0178] In some embodiments, the methods described herein comprise detecting an amount of expression of Z.PRCAT47 gene. In some embodiments, the PRCAT47gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for PRCAT47 is 53032. In some embodiments, the PRCAT47 gene is located at chromosome position 16q23.2. In some embodiments, Z.PRCAT47 gene comprises the nucleotide sequence of Ensembl gene identifierENSG00000260896. In some embodiments, a PRCAT47 gene comprises the nucleotide sequence of SEQ ID NO: 42 or a variant thereof.

[0179] In some embodiments, the methods described herein comprise detecting an amount of expression of a SPINK1 gene. In some embodiments, the SPINK1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for SPINK1 is 11244. In some embodiments, the SPINK1 gene is located at chromosome position 38.pl4. In some embodiments, a SPINK1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000164266. In some embodiments, a SPINK1 gene comprises the nucleotide sequence of SEQ ID NO:43 or a variant thereof.

[0180] In some embodiments, the methods described herein comprise detecting an amount of expression of a TDO2 gene. In some embodiments, the TDO2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TDO2 is 11708. In some embodiments, the TDO2 gene is located at chromosome position 4q32.1. In some embodiments, a TDO2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000151790. In some embodiments, an TDO2 gene comprises the nucleotide sequence of SEQ ID NO: 44 or a variant thereof.

[0181] In some embodiments, the methods described herein comprise detecting an amount of expression of a TK1 gene. In some embodiments, the TK1 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TK1 is 11830. In some embodiments, the TK1 gene is located at chromosome position 17q25.3. In some embodiments, a TK1 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000167900. In some embodiments, a TK1 gene comprises the nucleotide sequence of SEQ ID NO:45 or a variant thereof.

[0182] In some embodiments, the methods described herein comprise detecting an amount of expression of a TMEFF2 gene. In some embodiments, the TMEFF2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for TMEFF2 is 11867. In some embodiments, the TMEFF2 gene is located at chromosome position 2q32.3. In some embodiments, a TMEFF2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000144339. In some embodiments, a TMEFF2 gene comprises the nucleotide sequence of SEQ ID NO:46 or a variant thereof.

[0183] In some embodiments, the methods described herein comprise detecting an amount of expression of a VSTM2L gene. In some embodiments, the VSTM2L gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for VSTM2L is 16096. In some embodiments, the VSTM2L gene is located at chromosome position 20ql 1.23. In someembodiments, a VSTM2L gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000132821. In some embodiments, a VSTM2L gene comprises the nucleotide sequence of SEQ ID NO:47 or a variant thereof.

[0184] In some embodiments, the methods described herein comprise detecting an amount of expression of a KLK2 gene. In some embodiments, the KLK2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for KLK2 is 6363. In some embodiments, the KLK2 gene is located at chromosome position 19q 13.33. In some embodiments, KLK2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000167751. In some embodiments, KLK2 gene comprises the nucleotide sequence of SEQ ID NO: 48 or a variant thereof.

[0185] In some embodiments, the methods described herein comprise detecting an amount of expression of NUDT8 gene. In some embodiments, the NUDT8 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for NUDT8 is 8055. In some embodiments, the NUDT8 gene is located at chromosome position 11 ql 3.2. In some embodiments, NUDT8 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000167799. In some embodiments, NUDT8 gene comprises the nucleotide sequence of SEQ ID NO:49 or a variant thereof.

[0186] In some embodiments, the methods described herein comprise detecting an amount of expression of an EEF1A2 gene. In some embodiments, the EEF1A2 gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for EEF1A2 is 3192. In some embodiments, the EEF1A2 gene is located at chromosome position 20ql3.33. In some embodiments, an EE 1A2 gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000101210. In some embodiments, an EEF1A2 gene comprises the nucleotide sequence of SEQ ID NO: 50 or a variant thereof.

[0187] In some embodiments, the methods described herein comprise detecting an amount of expression of an SPDEF gene. In some embodiments, the SPDEF gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for SPDEF is 17257. In some embodiments, the SPDEF gene is located at chromosome position 6p21.31. In some embodiments, an SPDEF gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000124664. In some embodiments, an SPDEF gene comprises the nucleotide sequence of SEQ ID NO: 51 or a variant thereof.

[0188] In some embodiments, the methods described herein comprise detecting an amount of expression of a GAPDH gene. In some embodiments, the GAPDH gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for GAPDH is 4141. Insome embodiments, the GAPDH gene is located at chromosome position 12pl3.31. In some embodiments, a GAPDH gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000111640. In some embodiments, a GAPDH gene comprises the nucleotide sequence of SEQ ID NO: 52 or a variant thereof.

[0189] In some embodiments, the methods described herein comprise detecting an amount of expression of a LBH gene. In some embodiments, the LBH gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for LBH s 29532. In some embodiments, the LBH gene is located at chromosome position 2p23.1. In some embodiments, a LBH gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000213626. In some embodiments, a LBH gene comprises the nucleotide sequence of SEQ ID NO:53 or a variant thereof.

[0190] In some embodiments, the methods described herein comprise detecting an amount of expression of an HPN gene. In some embodiments, the HPN gene comprises the nucleotide sequence provided by HGNC. In some embodiments, the HGNC identifier for HPNis 5155. In some embodiments, the HPN gene is located at chromosome position 19ql3.11. In some embodiments, an HPN gene comprises the nucleotide sequence of Ensembl gene identifier ENSG00000105707. In some embodiments, an HPN gene comprises the nucleotide sequence of SEQ ID NO:54 or a variant thereof.

[0191] Sequence(s) of a subject’s gene disclosed herein are available from publicly available gene sequence databases, including but not limited to the Ensembl Genome Browser (available at https: / / www.ensembl.org / index.html).

[0192] Nucleotide sequences of illustrative genes of the subject are provided in Table 1.Table 1. Illustrative nucleotide sequences of genes of the subject.

[0193] In some embodiments, the methods described herein comprise detecting an amount of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 of genes ACSM1, AMACR, AR, COL9A2, CRISP3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRJN3A,RINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDRIM5, PEX10, PI A l A, PRA2G7, PRCA T47, SPINK1, 11)02, TKI, IMEFF2, TRGV9.1, VSTM2R, TMPRSS2-ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SP0N2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, H0XC6, and KLK3.

[0194] In some embodiments, the methods described herein comprise detecting an amount of expression of each of genes ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DRX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRIN3A, LINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PLA2G7, PRCAT47, SPINK1, TDO2, TKI, TMEFF2, TRGV9.1, VSIM2L, TMPRSS2-ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, H0XC6, and KI.K3.

[0195] In some embodiments, detecting an amount of expression of one or more of genes ACSM1, AMACR, AR, COL9A2, CRISP3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0EM1, GRIN3A, LINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PRA2G7, PRCAT47, SPINK1, TDO2, TKI, IMEFF2, TRGV9.1, VSRM2R, RMPRSS2-ERG, SCHRAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KEK4, HOXC6, and KRK3 comprises detecting an RNA expressed by the one or more of the genes or an amount of the RNA’s expression. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is IncRNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0196] In some embodiments, detecting an amount of expression of one or more of genes ACSM1, AMACR, AR, COR9A2, CRISP3, CST2, DRXI, ERV1, F5, GDF15, GRYATR1, G0EM1, GRRN3A, RINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDRIM5, PEX10, PRAIA, PRA2G7, PRCAT47, SPINK1, TDO2, TKI, IMEFF2, TRGV9.1, VSTM2R, IMPRSS2-ERG, SCHRAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KEK4, HOXC6, and KRK3 comprises detecting a cDNA reverse transcribed from an RNA expressed by the one or more of the genes or an amount of the cDNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is IncRNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0197] In some embodiments, the methods described herein comprise detecting an amount of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of genes TMPRSS2-ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KRK4, and H0XC6.

[0198] In some embodiments, the methods described herein comprise detecting an amount of expression of each of TMPRSS2-ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, RMSB15A, ERG, KRK4, and H0XC6.

[0199] In some embodiments, detecting an amount of expression of one or more of genes IMPRSS2- ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, H0XC6, and KLK3 comprises detecting an mRNA or an amount of its expression.

[0200] In some embodiments, detecting an amount of expression of one or more of genes IMPRSS2- ERG, SCHLAP1, OR51E2, APOCI, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, HOXC6, and KLK3 comprises detecting a cDNA reverse transcribed from an mRNA of the one or more genes.

[0201] In some embodiments, the methods described herein comprise detecting an amount of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of genes ACSMI, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRRN3A, LINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PLA2G7, PRCAT47, SPINK1, TDO2, TKI, RMEFF2, TRGV9.1, and VSTM2L.

[0202] In some embodiments, the methods described herein comprise detecting an amount of expression of each of genes ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRIN3A, LINC00993, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PRA2G7, PRCAT47, SPINK1, TDO2, TKI, TMEFF2, TRGV9.1, and VSIM2R.

[0203] In some embodiments, detecting an amount of expression of one or more of genes ACSM1, AMACR, AR, COR9A2, CRISP3, CST2, DRX1, ERV1, F5, GDF15, GRYATR1, G0EM1, GRRN3A, RINC0093, RRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDRIM5, PEX10, PRAIA, PRA2G7, PRCAT47, SPINK1, TDO2, TK1, IMEFF2, TRGV9.1, and VSTM2R comprises detecting an RNA expressed by the one or more of the genes or an amount of the RNA’s expression In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is IncRNA. In some embodiments, the RNA is piRNA. In someembodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0204] In some embodiments, detecting an amount of expression of one or more of genes ACSM1, AMACR, AR, COL9A2, CRISP3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, GOLM1, GRJN3A, LINC0093, LRRN1, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PLA2G7, PRCAT47, SPINK1, TDO2, TK1, TMEFF2, TRGV9.1, and VSTM2L comprises detecting a cDNA reverse transcribed from an RNA expressed by the one or more of the genes or an amount of the cDNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is IncRNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0205] In some embodiments, the gene is a kidney cancer gene. Illustrative kidney cancer genes are described in Table 2 below.Table 2.

[0206] In some embodiments, the kidney cancer gene is BMPR1B, CDH2, COL7A1, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, or SOD2.

[0207] In some embodiments, the methods described herein comprise detecting an amount of expression of one or more of genes BMPR1B, CDH2, COIF Al, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2.

[0208] In some embodiments, the methods described herein comprise detecting an amount of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of genes BMPR1B, CDH2, COL7A1, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2.

[0209] In some embodiments, the methods described herein comprise detecting an amount of expression of each of genes BMPR1B, CDH2, COL7A1, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2.

[0210] In some embodiments, detecting an amount of expression of one or more of genes BMPR1B, CDH2, COL7A1, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2 comprises detecting an RNA expressed by the one or more of the genes or an amount of the RNA’s expression. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In someembodiments, the RNA is long non-coding RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0211] In some embodiments, detecting an amount of expression of one or more of genes BMPR1B. CDH2, COL7A1, FGFR2, HDHD3, ICAM1, .JCAl), PFKFB4, MIR15A, MIR150, EG FIR MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2 comprises detecting cDNA reversed transcribed from an RNA expressed by the one or more of the genes or an amount of the cDNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is long non-coding RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0212] In some embodiments, the gene is a bladder cancer gene. Illustrative bladder cancer genes are described in Table 3 below.Table 3.

[0213] In some embodiments, the bladder cancer gene isABLl, ANXA10, UPK1B, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SI.C1A6, XIAP, CDKI, PRSSI, (ASP 14, KLHDC7B, ANRIL, MKLN1-AS, TALAM1, TTN-AS1, UCA1, LNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MALAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86-AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, or GLI3.

[0214] In some embodiments, the methods described herein comprise detecting an amount of expression of one or more of genes ABL1, ANXA10, UPK1B, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SLC1A6, XIAP, CDKI, PRSSI, CASP14, KLHDC7B, ANRIL, MKLN1-AS, TATAMI, TTN-AS1, UCA1, LNMAT2,BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MALAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86- AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, and GLI3.

[0215] In some embodiments, the methods described herein comprise detecting an amount of expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 of genes ABU, ANXA10, UPK1B, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SLC1A6, XIAP, CDK1, PRSSI, CASP14, KLHDC7B, ANRIL, MKTN1-AS, TATAMI, TTN-AS1, UCA1, LNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MALAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86- AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, and GLI3.

[0216] In some embodiments, the methods described herein comprise detecting an amount of expression of each of genes ABU, ANXA10, UPK1B, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SLC1A6, XIAP, CDK1, PRSSI, CASP14, KLHDC7B, ANRIL, MKLN1-AS, TATAMI, TTN-AS1, UCA1, TNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MATAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86- AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, and GTI3.

[0217] In some embodiments, detecting an amount of expression of one or more of genes ABT1, ANXA10, UPK1B, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, TCN2, MAGEA3, RPS21, STC1A6, XIAP, CDK1, PRSSI, CASP14, KLHDC7B, ANRIT,MKLN1-AS, TATAMI, TTN-AS1, UCA1, TNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MATAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86-AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, and GTI3 comprises detecting an RNA expressed by the one or more of the genes or an amount of the RNA’s expression. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is micro-RNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is long non-coding RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In someembodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0218] In some embodiments, detecting an amount of expression of one or more of genes ABL1, ANXAIO, UPKIB, CRH, IGF2, HOXA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SLC1A6, XIAP, CDK1, PRSSI, CASPI4, KLHDC7B, ANRIL, MKLN1-AS, TALAM1, TTN-AS1, UCA1, LNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MALAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86-AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, and GLI3 comprises detecting cDNA reversed transcribed from an RNA expressed by the one or more of the genes or an amount of the cDNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is micro-RNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is a long non-coding RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0219] In some embodiments, the gene is a gene of a pathogen, e.g., a sexually transmitted infection (STI) pathogen or a urinary tract infection (UTI) pathogen. In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is the bacterium is of the genus Escherichia, Klebsiella, Proteus, Enterococcus, Staphylococcus, Pseudomonas, Staphylococcus, Streptococcus, or Staphylococcus. In some embodiments, the bacterium is Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Streptococcus agalactiae or Staphylococcus aureus.

[0220] In some embodiments, the pathogen is a yeast. In some embodiments, the yeast is of the genus Candida. In some embodiments, the yeast is Candida albicans.

[0221] In some embodiments, the gene is a gene of a pathogen and the gene is 16S rRNA. In some embodiments, the gene is of a pathogen and the gene is 23S rRNA. In some embodiments, the gene is of a pathogen and the gene is 26S rRNA.

[0222] In some embodiments, the gene is from the subject’s immune response to the pathogen, e.g., a gene of the subject’s B cell or T cell engaged in the immune response.Detecting Expression of Genes

[0223] In some embodiments, detecting the extracted RNA comprises detecting an amount of expression of the subject’s gene. In some embodiments, detecting the extracted RNA comprisesdetecting an amount of expression of RNA transcribed from the subject’s gene. In some embodiments, detecting the extracted RNA comprises detecting the amount of mRNA transcribed from the gene. In some embodiments, detecting the extracted RNA comprises reverse transcribing the extracted RNA to complementary DNA (cDNA) using reverse transcriptase and detecting the cDNA.

[0224] The amount of expression of a subject’s gene can be detected using any of a variety of nucleic acid techniques. Suitable techniques include, but are not limited to: nucleic acid sequencing; nucleic acid hybridization; and nucleic acid amplification. In some embodiments, detecting the extracted RNA comprises performing: reverse transcription-quantitative polymerase chain reaction (RT-qPCR); RNA-sequencing; reverse transcription digital PCR (RT-dPCR); quantitative PCR (qPCR); digital PCR (dPCR); microarray analysis; Northern Blot; UV spectroscopy; or fluorometry.

[0225] The amount of gene expression can be detected using a Second Generation (z.e., Next Generation or Next-Gen), Third Generation (z.e., Next-Next-Gen), or Fourth Generation (z.e., N3- Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008). Those of skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack, the extracted RNA can be reverse transcribed to complementary DNA (cDNA) before sequencing. A number of DNA sequencing techniques are suitable for gene expression detection, including fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y. In some embodiments, the sequencing is automated sequencing techniques understood in the art. In some embodiments, the sequencing is parallel sequencing of partitioned amplicons (PCT Publication No: W02006084132 to Kevin McKeman et al. In some embodiments, the sequencing is DNA sequencing by parallel oligonucleotide extension (See, e.g., U.S. Patent No. 5,750,341 to Macevicz et al., and U.S. Patent No. 6,306,597 to Macevicz et al. Additional examples of sequencing techniques include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Patent No. 6,432,360, U.S. Patent No. 6,485,944, U.S. Patent No. 6,511,803, the 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; US 20050130173, the Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Patent No. 6,787,308; U.S. Patent No. 6,833,246, the Lynx massively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Patent No. 5,695,934; U.S. Patent No. 5,714,330, and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 00018957.

[0226] Illustrative non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot.

[0227] In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH can be used to measure and localize mRNAs and other transcripts (e.g., cancer markers) within tissue sections or whole mounts. Sample cells and tissues can be treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

[0228] Expression of each of the one or more genes of the present methods can be detected by conducting one or more hybridization reactions. The one or more hybridization reactions can comprise one or more hybridization arrays, hybridization reactions, hybridization chain reactions, isothermal hybridization reactions, nucleic acid hybridization reactions, or a combination thereof. The one or more hybridization arrays can comprise hybridization array genotyping, hybridization array proportional sensing, DNA hybridization arrays, macroarrays, microarrays, high-density oligonucleotide arrays, genomic hybridization arrays, comparative hybridization arrays, or a combination thereof.

[0229] Microarrays including, but not limited to, DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays); protein microarrays; tissue microarrays; transfection or cell microarrays; chemical compound microarrays; and antibody microarrays, can optionally be employed. A DNA microarray, commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g., glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously. The affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify disease genes or transcripts by comparing gene expression in diseased and normal cells. Microarrays can be fabricated using a variety of technologies, including but not limited to: printing with fine-pointedpins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.

[0230] Detection of an amount of expression of the one or more genes of the present methods can comprise conducting one or more amplification reactions. Nucleic acids can be amplified prior to or simultaneous with detection. Conducting one or more amplification reactions can comprise one or more PCR-based amplifications, non-PCR based amplifications, or a combination thereof.Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR), reverse transcript! on-polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiple displacement amplification (MDA), real-time SDA, rolling circle amplification, circle-to-circle amplification transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g, PCR) require that RNA be reversed transcribed to complementary DNA (cDNA) prior to amplification by qPCR or dPCR , whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[0231] The polymerase chain reaction (U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called reverse transcription-polymerase chain reaction (RT- PCR), reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. In some embodiments, the RT-PCR is RT-dPCR. In some embodiments, the RT-PCR is RT-qPCR. For other various permutations of PCR ee, e.g, U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155: 335 (1987); and Murakawa etal., DNA 7: 287 (1988). In some embodiments, the reverse transcriptase is Moloney murine leukemia virus (M-MLV) reverse transcriptase.

[0232] Transcription mediated amplification (U.S. Patent Nos. 5,480,784 and 5,399,491), commonly referred to as TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies. See, e.g., U.S. Patent Nos. 5,399,491 and 5,824,518. In a variation described in U.S. Publ. No. 20060046265, TMA optionally incorporates the use of blocking moi eties, terminating moi eties, and other modifying moieties to improve TMA process sensitivity and accuracy.

[0233] The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid. The DNA oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.

[0234] Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Patent Nos. 5,270,184 and 5,455,166), commonly referred to as SDA, uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPaS to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3' end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Patent No. 0 684 315).

[0235] Other amplification methods include, for example: nucleic acid sequence-based amplification (U.S. Patent No. 5,130,238), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi et al., BioTechnoL 6: 1197 (1988)), commonly referred to as QP replicase; a transcription-based amplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989)); and, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874 (1990)). For further discussion of known amplification methods see Persing, David H., “In Vitro Nucleic Acid Amplification Techniques” in Diagnostic Medical Microbiology: Principles and Applications (Persing et al., Eds.), pp. 51-87 (American Society for Microbiology, Washington, DC (1993)).

[0236] In some embodiments, amplification methods are quantitative PCR methods (qPCR), also known as real time PCR (RT-PCR). qPCR is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR. qPCR can be used quantitatively or semi-quantitatively (i.e., above / below a certain amount of DNA molecules). Two common methods for the detection of PCR products in qPCR are (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence.

[0237] In some embodiments, detecting an amount of gene expression comprises detecting RNA or an amount of its expression. In some embodiments, the amount of RNA is detected by reversetranscribing the RNA to cDNA and detecting the cDNA using qPCR analysis, which provides a Crt (cycle threshold) value for each cDNA detected. In a qPCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Crt value is defined as the number of cycles required for the fluorescent signal to cross the threshold (z.e., to exceed the background level). Crt values are inversely proportional to the amount of target nucleic acid in the sample (z.e., the lower the Crt value the greater the amount of mRNA in the sample). In some embodiments, the Crt value is about 35 cycles, about 34 cycles, about 33 cycles, about 32 cycles, about 31 cycles, about 30 cycles, about 29 cycles, about 28 cycles, about 27 cycles, about 26 cycles, about 25 cycles, about 24 cycles, about 23 cycles, about 22 cycles, about 21 cycles, about 20 cycles, about 19 cycles, about 18 cycles, about 17 cycles, about 16 cycles, about 15 cycles, or lower using the RNA extraction methods described herein. In some embodiments, the Crt value is about 35 cycles or lower using the RNA extraction methods described herein. In some embodiments, the Crt value is about 30 cycles or lower. In some embodiments, the Crt value is about 25 cycles or lower. In some embodiments, the Crt value is about 20 cycles or lower. In some embodiments, the gene is one or more of the of the prostate cancer, kidney cancer, bladder cancer, STI, UTI or yeast genes disclosed herein. In some embodiments, the gene is 16S rRNA, 23S rRNA or 26S rRNA. In some embodiments, the gene is of the subject’s B cell or T cell engaged in an immune response.

[0238] In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is long non-coding RNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0239] In some embodiments, the amount of extracted RNA or cDNA detectable according to the RNA extraction methods described herein is significantly greater than that detectable from a method that does not comprise admixing whole urine and a solid support comprising silicon carbide. In some embodiments, the amount of extracted RNA or cDNA detectable according to the RNA extraction methods described herein about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 75-fold, or about 100-fold greater than the amount of extracted RNA or cDNA detectable according to a method that does not comprise admixing whole urine and a solid support comprising silicon carbide. In some embodiments, the amount of extracted RNA or cDNA detectable according to the RNA extraction methods described herein is about 100%, about 150%, about 175%, about 200%, about 250%, about 275%, about 300%, about 350%, about 375%, about 400%, about 450%, about 475%, about 500%,about 550%, about 575%, about 600%, about 650%, about 675%, about 700%, about 800%, about 900%, or about 1000% greater than the amount of extracted RNA or cDNA detectable according to a method that does not comprise admixing whole urine and a solid support comprising silicon carbide.

[0240] In some embodiments, RT-qPCR comprises transcribing cDNA from extracted RNA using a Moloney murine leukemia virus (M-MLV) reverse transcriptase. In some embodiments, the ratio of M-MLV reverse transcriptase to extracted RNA to elution buffer is about 1 :about 11 :about 100 (v / v / v), for example, about 1 pL of M-MLV reverse transcriptase to about 11 pL of extracted RNA in about 100 pL of elution buffer.

[0241] In some embodiments, the amount of expression of any one of the genes described herein is normalized to an amount of expression of a reference gene. In some embodiments, the amount of expression of mRNA is normalized to an amount of expression of mRNA of a reference gene. Reference genes suitable for normalization are known to those of skill in the art and include, but are not limited to, KLK3, CYPB561A3, EEF1A2, GAPDH, HPN, KLK2, LBH, NUDT8, SPDEF, or TRGV. In some embodiments, the reference gene is KLK3.

[0242] Compositions that are useful for detecting an amount of gene expression (“detection compositions”) can comprise one or more antibodies, probes, amplification oligonucleotides or reagents.

[0243] Detection compositions can comprise 1 or more, 2 or more, 3 or more, or 4 or more antibodies, probes, pairs of probes, pairs of amplification oligonucleotide, or sequencing primers. The probes or primers can hybridize to 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, or 21 or more target molecules. The target molecules may be RNA, DNA, cDNA, mRNA, miRNA, siRNA, long non-coding RNA, circular RNA, piRNA, rRNA, tRNA, hnRNA or ncRNA; a portion or fragment thereof; or a combination thereof. In some instances, at least a portion of the target molecules are cancer markers. The probes may hybridize to 1 or more, or 2 or more cancer markers disclosed herein. Typically, the probes or primers comprise a target specific sequence. The target specific sequence may be complementary to at least a portion of the target molecule. The target specific sequence may be at least about 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 100% complementary to at least a portion of the target molecule. The target specific sequence can be at least about 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more nucleotides in length. In some instances, thetarget specific sequence is between about 8 to about 20 nucleotides, 10 to about 18 nucleotides, or 12 to about 16 nucleotides in length.

[0244] These detection compositions can comprise a plurality of probes or primers, wherein the two or more probes of the plurality of probes comprise identical target specific sequences. The detection compositions may comprise a plurality of probes, wherein the two or more probes of the plurality of probes comprise different target specific sequences.

[0245] The probes can further comprise a unique sequence. The unique sequence is noncomplementary to the target marker. The unique sequence may comprise a label, barcode, or unique identifier. The unique sequence may comprise a random sequence, nonrandom sequence, or a combination thereof. The unique sequence may be at least about 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more nucleotides in length. In some instances, the unique sequence is between about 8 to about 20 nucleotides, 10 to about 18 nucleotides, or 12 to about 16 nucleotides in length.

[0246] The probes can further comprise a universal sequence. The universal sequence may comprise a primer binding site. The universal sequence may enable detection of the target sequence. The universal sequence may enable amplification of the target sequence. The universal sequence may enable transcription or reverse transcription of the target sequence. The universal sequence may enable sequencing of the target sequence.

[0247] Detection compositions, such as for RNA-sequencing, comprising a probe or primer can be provided on a solid support. The solid support can comprise one or more beads, plates, solid surfaces, wells, chips, or a combination thereof. The beads can be magnetic, antibody coated, protein A crosslinked, protein G crosslinked, streptavidin coated, oligonucleotide conjugated, silica coated, or a combination thereof. Examples of beads include, but are not limited to, Ampure beads, AMPure XP beads, streptavidin beads, agarose beads, magnetic beads, DYNABEADS, MACS microbeads, antibody conjugated beads (e.g.,, anti-immunoglobulin microbead), protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo-dT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and BCMAG Carboxy-Terminated Magnetic Beads.

[0248] The detection compositions can comprise one or more primers or primer pairs capable of amplifying target molecules, or fragments or subsequences or complements thereof. The nucleotide sequences of the target molecules may be provided in computer-readable media for in silico applications and as a basis for the design of appropriate primers for amplification of one or more target molecules.I l l

[0249] Primers based on the nucleotide sequences of target molecules can be designed for use in amplification of the target molecules. For use in amplification reactions such as PCR, a pair of primers can be used. The exact composition of the primer sequences is not critical to the disclosure, but for most applications the primers may hybridize to specific sequences of the target molecules or the universal sequence of the probe under stringent conditions, particularly under conditions of high stringency, as known in the art. The pairs of primers are usually chosen so as to generate an amplification product of at least about 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more nucleotides. Algorithms for the selection of primer sequences are generally known and are commercially available. These primers may be used in standard quantitative or qualitative PCR-based assays to assess transcript expression levels of target molecules. Alternatively, these primers may be used in combination with probes, such as molecular beacons in amplifications using qPCR.

[0250] The nucleotide sequence of the entire length of the primer does not need to be derived from the target sequence. Thus, for example, the primer may comprise nucleotide sequences at the 5’ and / or 3’ termini that are not derived from the target molecule. Nucleotide sequences which are not derived from the nucleotide sequence of the target molecule may provide additional functionality to the primer. For example, they may provide a restriction enzyme recognition sequence or a “tag” that facilitates detection, isolation, purification or immobilization onto a solid support. Alternatively, the additional nucleotides may provide a self-complementary sequence that allows the primer to adopt a hairpin configuration. Such configurations may be necessary for certain primers, for example, molecular beacon and Scorpion primers, which can be used in solution hybridization techniques.

[0251] The probes or primers can incorporate moieties useful in detection, isolation, purification, or immobilization, if desired. Such moieties are well-known in the art (see, for example, Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology, Wiley & Sons, New York) and are chosen such that the ability of the probe to hybridize with its target molecule is not affected.

[0252] Examples of suitable moieties are detectable labels, such as radioisotopes, fhiorophores, chemiluminophores, enzymes, colloidal particles, and fluorescent microparticles, as well as antigens, antibodies, haptens, avidin / streptavidin, biotin, haptens, enzyme cofactors / substrates, enzymes, and the like.

[0253] A label can optionally be attached to or incorporated into a probe or primer to allow detection and / or quantitation of a target polynucleotide representing the target molecule of interest. The target polynucleotide may be the expressed target molecule RNA itself, a cDNA copy thereof, or anamplification product derived therefrom, and may be the positive or negative strand, so long as it can be specifically detected in the assay being used. Similarly, an antibody may be labeled.

[0254] In certain multiplex formats, labels used for detecting different target molecules may be distinguishable. The label can be attached directly (e.g., via covalent linkage) or indirectly, e.g., via a bridging molecule or series of molecules (e.g., a molecule or complex that can bind to an assay component, or via members of a binding pair that can be incorporated into assay components, e.g., biotin-avidin or streptavidin). Many labels are commercially available in activated forms which can readily be used for such conjugation (for example through amine acylation), or labels may be attached through known or determinable conjugation schemes, many of which are known in the art.

[0255] Labels useful in the disclosure described herein include any substance which can be detected when bound to or incorporated into the target molecule. Any effective detection method can be used, including optical, spectroscopic, electrical, piezoelectrical, magnetic, Raman scattering, surface plasmon resonance, colorimetric, calorimetric, etc. A label is typically selected from a chromophore, a lumiphore, a fluorophore, one member of a quenching system, a chromogen, a hapten, an antigen, a magnetic particle, a material exhibiting nonlinear optics, a semiconductor nanocrystal, a metal nanoparticle, an enzyme, an antibody or binding portion or equivalent thereof, an aptamer, and one member of a binding pair, and combinations thereof. Quenching schemes may be used, wherein a quencher and a fluorophore as members of a quenching pair may be used on a probe, such that a change in optical parameters occurs upon binding to the target introduce or quench the signal from the fluorophore. One example of such a system is a molecular beacon. Suitable quencher / fluorophore systems are known in the art. The label may be bound through a variety of intermediate linkages. For example, a target polynucleotide may comprise a biotin-binding species, and an optically detectable label may be conjugated to biotin and then bound to the labeled target polynucleotide. Similarly, a polynucleotide sensor may comprise an immunological species such as an antibody or fragment, and a secondary antibody containing an optically detectable label may be added.

[0256] Chromophores useful in the methods described herein include any substance which can absorb energy and emit light. For multiplexed assays, a plurality of different signaling chromophores can be used with detectably different emission spectra. The chromophore can be a lumophore or a fluorophore. Typical fluorophores include fluorescent dyes, semiconductor nanocrystals, lanthanide chelates, polynucleotide-specific dyes (e.g., intercalating dyes) and fluorescent proteins (e.g., GFP or RFP).Subjects, Samples, and Kits

[0257] In some embodiments, the present methods comprise extracting RNA present in whole urine from a subject’s urine sample. In some embodiments, the present methods comprise detecting anamount of expression of RNA extracted from whole urine from a subject’s urine sample. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is long non-coding RNA. In some emobimdents, the RNA is circular RNA. In some embodiments, the RNA is piRNA. In some embodiments, the RNA is rRNA. In some embodiments, the RNA is tRNA. In some embodiments, the RNA is hnRNA. In some embodiments, the RNA is ncRNA.

[0258] In some embodiments, the whole urine comprises first-catch urine. In some embodiments, the whole urine is first-catch urine.

[0259] The methods disclosed herein are useful for both male and female subjects where the gene is not a prostate cancer gene. In some embodiments, the subject is a human subject.

[0260] In some embodiments, the subject is male. In some embodiments, the male subject has a prostate. In some embodiments, the subject is prostate biopsy-naive. In some embodiments, the subject is prostate biopsy-prior negative. In some embodiments, the subject is prostate biopsy-prior negative for Grade Group >2 prostate cancer. In some embodiments, the subject is a male subject, and the urine sample provided by the subject comprises prostatic fluid. In some embodiments, the subject is a male human having a prostate.

[0261] In some embodiments, a subject from whom a urine sample is obtained is selected by a skilled practitioner, e.g., internist, urologist or oncologist.

[0262] In some embodiments, the subject had a DRE within about 180 minutes before providing the urine sample. In some embodiments, the subject is a male human having a prostate and the male human having a prostate had a DRE within about 180 minutes before providing the urine sample.

[0263] In some embodiments, the subject did not have a DRE within about 180 minutes before providing the urine sample. As a DRE is invasive and uncomfortable, it is believed that the present methods’ use of a urine sample provided by a subject that did not have a DRE within about 180 minutes before providing the urine sample can result in higher subject compliance with urine-based tests for prognosis and / or diagnosis, such as for prostate cancer prognosis and / or diagnosis. In some embodiments, the subject is a male human having a prostate and the male human having a prostate did not have a DRE within about 180 minutes before providing a urine sample.

[0264] In some embodiments, the subject has or is suspected of having cancer. In some embodiments, the subject has or is suspected of having prostate cancer. In some embodiments, the prostate cancer is Grade Group (GG) >2 prostate cancer. In some embodiments, the prostate cancer is GG >3 prostate cancer. In some embodiments, the prostate cancer is GG >4 prostate cancer. In some embodiments, the prostate cancer is GG5 prostate cancer. In some embodiments, the prostate cancer has metastasized.

[0265] In some embodiments, the urine sample provided by a subject is provided outside a clinical setting. In some embodiments, the urine sample is provided directly by the subject, z.e., without the assistance of a clinician or medical service provider.

[0266] In some embodiments, the urine for use in the present methods comprises a nucleic acid, and the detecting comprises detecting an amount of the nucleic acid. In some embodiments, the urine for use in the present methods comprises RNA, and the detecting comprises detecting an amount of the RNA. In some embodiments, the urine for use in the present methods comprises mRNA, and the detecting comprises detecting an amount of the mRNA or cDNA reversed transcribed from the mRNA. In some embodiments, the urine for use in the present methods comprises miRNA, and the detecting comprises detecting an amount of the miRNA or cDNA reversed transcribed from the miRNA. In some embodiments, the urine for use in the present methods comprises siRNA, and the detecting comprises detecting an amount of the siRNA or cDNA reverse transcribed from the siRNA. In some embodiments, the urine for use in the present methods comprises circular RNA, and the detecting comprises detecting an amount of the circular RNA or cDNA reversed transcribed from the circular RNA. In some embodiments, the urine for use in the present methods comprises long non-coding RNA, and the detecting comprises detecting an amount of the long non-coding RNA or cDNA reversed transcribed from the long non-coding RNA. In some embodiments, the urine for use in the present methods comprises piRNA, and the detecting comprises detecting an amount of the piRNA or cDNA reversed transcribed from the piRNA. In some embodiments, the urine for use in the present methods comprises rRNA, and the detecting comprises detecting an amount of the rRNA or cDNA reversed transcribed from the rRNA. In some embodiments, the urine for use in the present methods comprises tRNA, and the detecting comprises detecting an amount of the tRNA or cDNA reversed transcribed from the tRNA. In some embodiments, the urine for use in the present methods comprises hnRNA, and the detecting comprises detecting an amount of the hnRNA or cDNA reversed transcribed from the hnRNA. In some embodiments, the urine for use in the present methods comprises ncRNA, and the detecting comprises detecting an amount of the ncRNA or cDNA reverse transcribed from the ncRNA. In some embodiments, the urine is whole urine.

[0267] Further provided herein are kits for use in the present methods. In some embodiments, the kits comprise a container for containing the urine sample, and instructions for providing the urine sample. In some embodiments, the container is a sealable container, e.g., with a stoppered vial. In some embodiments, the container is commercially available from DNA Genotek Inc. under the trademark Colli-Pee.

[0268] In some embodiments, the container contains a buffer, e.g., for processing of the sample. In some embodiments, the buffer is a urine stabilization buffer. Buffers suitable for sample stabilizationare known to those of skill in the art and can be determined based on the type of sample being collected. In some embodiments, the buffer further comprises a preservative for adequate stability of the sample. In some embodiments, the buffer to sample ratio is 1 :4. In some embodiments, the buffer to sample ratio is 1 :4, 1 :2, or 3 :4. It will be understood that the container will be large enough to accommodate the sample, e.g., the urine sample, and the buffer.

[0269] In some embodiments, the container can contain a volume of about 1 mL to about 50 mL of urine sample. In some embodiments, the container can contain a volume of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL of urine sample.

[0270] In some embodiments, the container contains a preservative. In some embodiments, where the container contains a preservative and urine sample, the preservative to urine sample ratio is about 1 : 1 (v / v), about 1 :2 (v / v), about 1 :3 (v / v), about 1 :4 (v / v), about 1 :5 (v / v), about 2:5 (v / v), about 1 :6 (v / v), about 1 :7 (v / v), about 1 :8 (v / v), about 1 :9 (v / v), or about 1 : 10 (v / v). In some embodiments, the preservative to urine sample ratio is about 1 :3 (v / v). In some embodiments, the preservative to urine sample ratio is about 2:5 (v / v). In some embodiments, the preservative is Urine Conservation Medium (UCM). In some embodiments, the preservative is Urinary Analyte Stabilizer (UAS).

[0271] The urine sample can be stored, e.g., at low temperature, prior to use in the present methods. In some embodiments, the urine sample can be stored for two weeks or more at a temperature of about -80 °C to about -40 °C prior to use in the present methods. In some embodiments, the urine sample can be stored for two weeks or more at a temperature of about -80 °C or about -40 °C prior to use in the present methods. In some embodiments, the urine sample can be stored for fewer than two weeks at a temperature of about 4 °C prior to use in the present methods. In some embodiments, the urine sample can be stored for about one week at a temperature of about 23 °C prior to use in the present methods.

[0272] The urine sample for use in the present methods may be an archival sample, having a known and documented medical outcome, or may be a urine sample from a patient whose ultimate medical outcome is not yet known.

[0273] The present methods provide a significantly, surprisingly and unexpectedly greater extraction performance, e.g., as measured by cycle relative threshold (Crt), or a significantly, surprisingly and unexpectedly greater amount of extracted RNA, compared to that provided using a standard method for RNA extraction (e.g., the Thermo Fisher method or Norgen method described herein).EXAMPLES

[0274] The following Examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.Example la

[0275] RNA was extracted using reagents from the Urine Total RNA Purification Maxi Kit (High Throughput Maxi Slurry Format) (Norgen Biotech, catalog number 29650).

[0276] A sample of about 5 mL of whole urine from a urine sample from a male human subject having a prostate and who had a digital rectal examination (DRE) within about 180 minutes before providing the sample was aliquoted into a conical tube. About 0.35 mL of Slurry C3 (Norgen) and 4.65 mL of Lysis Buffer A (Norgen) were added to the urine. Cells within the urine were lysed by vortexing. About 5 mL of absolute ethanol was then added to the lysate and vortexed. The resultant mixture was then centrifuged for 5 min at 2,000 x g, and the supernatant was removed and discarded, leaving the pellet.

[0277] 500 pL Wash Solution A (Norgen) was then added to the pellet and admixed by pipetting or vortexing. A 96-Well Filter Plate (Norgen) was placed on top of a 96-Well Collection Plate (Norgen), and the pellet admixture was pipetted to a well of the 96-Well Filter Plate. The resultant assembly was then centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes, and flowthrough was removed and discarded.

[0278] 400 pL of Wash Solution (Norgen) was added to the columns of the Filter Plate and centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes, and flowthrough was removed and discarded. The 96-well filter plate was then placed on a new 96-well PCR plate.

[0279] For every on-column reaction to be performed, an admixture of 15 pL of DNase I (Norgen) and 100 pL of Enzyme Incubation Buffer was prepared by gently mixing. 115 pL of DNase I (Norgen) and Enzyme Incubation Buffer (Norgen) was added to the Filter Plate column and centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes. The 96-well Filter plate was placed back on top of the 96-Well Collection plate. The flowthrough present in the 96-well PCR plate was then placed back onto the top of the column and incubated at room temperature for 15 minutes.

[0280] 500 pL of Wash Solution A (Norgen) was added to each used well of the 96-Well Filter Plate and centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes. The flowthrough was removed and discarded. The wash was repeated.

[0281] The bottom of the 96-Well Filter Plate was patted dry, and the 96-Well Filter Plate and the collection plate were reassembled. The assembly was then centrifuged at 3,146 x g (or 3,900 rpm) for 15 minutes to completely dry the plate.

[0282] 100 pL of Elution Solution A (Norgen) was then added to each used well of the plate, and incubated at room temperature for 2 min. The assembly was then centrifuged at 500 rpm for 2 min, then at 3,000 x g for 2 min, to achieve extracted RNA.Example lb

[0283] The amount of cDNA reverse transcribed from RNA extracted according to the method of Example la was compared to that of an RNA extraction method using the MAGMAX MIRV ANA Total RNA Isolation Kit (Thermo Fisher Scientific, Cat No A27828) in accordance with the manufacturer’s instructions (referred to herein as the “Thermo Fisher extraction method,” available at http s : / / assets. thermofi sher.com / TF S-Assets%2FLSG%2Fmanuals%2FMAN0011139_A27828_magmax_rnaisolation_urine_ug.pdf). Extracted RNA from each extraction method was then processed using the protocol described in Example 2a, to amplify and measure prostate cancer biomarkers. Quantitative polymerase chain reaction (qPCR) of the housekeeping gene KLK3 was measured by relative cycle threshold (Crt) and compared between the two RNA extraction methods in 24 subject samples (FIG. 1). The qPCR was performed in triplicate for each gene target. The amount of cDNA reverse transcribed from RNA extracted according to the method of Example la was consistently greater than that reverse transcribed from the cDNA of the Thermo Fisher extraction method, with an average reduction in Crt of 2.2 cycles (Avg Crt: Example la = 19.9 vs Thermo Fisher extraction method = 22.1), which is equivalent to a 4.59 fold increase in RNA extraction efficiency, as shown in FIG. 1.Table 4. Comparison of target gene amplification using the Example la method or the ThermoFisher extraction methodExample 2a

[0284] Reverse transcription reaction mixtures were prepared using Takara PRIMESCRIPT RT reagent PRIMESCRIPT Buffer, random 6mers, and reverse transcriptase (RT) enzyme.

[0285] PRIMESCRIPT Buffer, random 6mers, and enzyme were admixed at a ratio of 4:4: 1 to generate a master mix. The master mix and extracted RNA (e.g., from Example la supra) were admixed at a ratio of 9: 11 (for a ratio of RT enzyme to RNA of 1 : 11). RNA was not normalized. Reaction mixtures were then mixed (e.g., vortexed) and spun down (e.g., by performing a centrifugation).

[0286] The following thermal cycling conditions were run: 37 °C for 15 minutes, 85 °C for 5 seconds, and hold at 4 °C.Example 2b

[0287] The reverse transcription efficiency of Example 2a was compared to that of a conventional reverse transcription method (the SUPERSCRIPT IV VILO Master Mix (Thermo Fisher Scientific, Cat No 11756050)), referred to herein as the “Thermo Fisher reverse transcription method”. Reverse transcribed complementary DNA (cDNA) from both methods was processed using the detection protocol of Example 2a to amplify and measure prostate cancer biomarkers. Quantitative polymerase chain reaction (qPCR) of the housekeeping gene KLK3 was measured by relative cycle threshold (Crt) and compared between the two reverse transcription methods across 96 samples. The RNA detection method of Example 2a consistently outperformed the Thermo Fisher reverse transcription method, with an average reduction in Crt of 0.67 cycles (Avg Crt: Example 2a = 15.20 vs Thermo Fisher reverse transcription method = 15.86), which is equivalent to a 1.59-fold increase in reverse transcription efficiency.Example 3

[0288] A multi-parameter experiment measured urine input volume, centrifugation time for drying, elution wash volume and final elution volume for RNA extraction from each of three pools (A, B and C) of clinical whole-urine remnants, z.e., whole urine pooled from two or more men having a prostate and who had a DRE within about 180 minutes before providing the urine (Table 2). RNA wasextracted as described in Example la supra, with the respective parameter modifications as detailed in Table 5: urine volume was 5 mL or 10 mL; drying spin time was 5 min, 10 min or 15 min, and elution 1 and 2 volume was 50 pL or 75 pL.Table 5. Parameters

[0289] RNA extraction performance was determined by measuring the relative cycle threshold (Crt) of cDNA reversed transcribed from extracted RNA expressed by KLK3 by qPCR as described in Example 2a supra. The amplification results are shown in Table 6. An “Undetermined” result designates no detectable amplification. From these results, the combination of parameters that resulted in the lowest KLK3 Crt, the threshold for cDNA reversed transcribed from extracted RNA expressed by KLK3, was a urine input of 5 mL, a drying spin time of 15 min and an Elution 1 and Elution 2 volume of 75 pL.Table 6. ResultsExample 4a

[0290] An RNA extraction procedure was performed according to Steps 1 to 25 described below. RNA extraction reagents for this procedure were obtained from the Urine Total RNA Purification Maxi Kit (Norgen Biotek Corp., High Throughput Maxi Slurry Format, Catalogue No. 29650) and the RNase-free DNase I Kit (Norgen Biotek Corp., Catalogue No. 25720).

[0291] Step 1. 5 mL of whole urine was aliquoted into a 50 mL conical tube. The whole urine was from a urine sample from a male human subject having a prostate who either had or did not have a digital rectal examination (DRE) within about 180 minutes before providing the sample.

[0292] Step 2. 0.35 mL Slurry C3 and 4.65 mL of Lysis Buffer A was added directly to the whole urine. Cells were lysed by vortexing for 15 seconds.

[0293] Step 3. 5 mL of absolute ethanol was added to the lysate product of Step 2 and mixed by vortexing for 10 seconds.

[0294] Step 4. The admixture was centrifuged for 5 min at 2,000 x g and 23 °C. The supernatant was discarded following centrifugation.

[0295] Step 5. 500 pL of Wash Solution A was added to the pellet and mixed by pipetting or vortexing.

[0296] Step 6. A 96-Well Filter Plate was placed on top of a provided 96-Well Collection Plate.

[0297] Step 7. The mixture from Step 5 was transferred into a well of the 96-Well Filter Plate.

[0298] Step 8. The assembly was centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes at 23 °C. The flowthrough was discarded following centrifugation. The 96-Well Filter Plate and the 96-Well Collection Plate were reassembled.

[0299] Step 9. 400 pL of Wash Solution (provided with Total RNA Purification Maxi Kit) was added to the well of the 96-Well Filter Plate and centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes at 23 °C. The flowthrough was discarded following centrifugation. The 96-Well Filter Plate was placed on a 96-well PCR plate (but the 96-Well Collection Plate was retained).

[0300] Step 10. A mix of 15 pL of DNase I and 100 pL of Enzyme Incubation Buffer was prepared in a tube. The tube was gently mixed by inverting a few times. The tube was not vortexed.

[0301] Step 11. 115 pL of DNase I / Enzyme Incubation Buffer admixture was added to the well of the 96-Well Filter Plate and centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes at 23 °C.

[0302] Step 12. The 96-well Filter Plate was placed on top of the 96-Well Collection Plate retained from Step 9.

[0303] Step 13. The flowthrough that was present in the well of the 96-well PCR plate was pipetted back into the well of the 96-Well Filter Plate.

[0304] Step 14. The 96-Well Filter Plate was incubated at room temperature for 15 minutes.

[0305] Step 15. 500 pL of Wash Solution A was added to the well of the 96-Well Filter Plate. The 96-Well Filter Plate and 96-Well Collection Plate assembly were centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes at 23 °C.

[0306] Step 16. The 96-Well Filter Plate and 96-Well Collection Plate were disassembled and the flowthrough was discarded. The 96-Well Filter Plate and 96-Well Collection Plate were reassembled.

[0307] Step 17. Steps 15 and 16 were repeated once.

[0308] Step 18. The bottom of the 96-Well Filter Plate was patted dry with a paper towel. The 96- Well Filter Plate and the 96-Well Collection Plate were reassembled.

[0309] Step 19. The 96-Well Filter Plate and the 96-Well Collection Plate were centrifuged at 3,146 x g (or 3,900 rpm) for 15 minutes at 23°C to completely dry the plate.

[0310] Step 20. 100 pL of Elution Solution A was added to each well of the 96-Well Filter Plate by pipetting the Elution Solution A down the side of the well slowly to soak the entire well.

[0311] Step 21. The 96-Well Filter Plate and the 96-Well Collection Plate assembly were incubated on the benchtop at room temperature for 2 minutes.

[0312] Step 23. The assembly was centrifuged at 500 RPM for 2 minutes at 23 °C.

[0313] Step 24. The assembly was centrifuged at 3,146 x g (or 3,900 rpm) for 2 minutes at 23 °C.

[0314] Step 25. The flowthrough was collected and placed in a tube to provide the extracted RNA. The tube was kept on ice or frozen at -80 °C until further analysis.Example 4b

[0315] A cDNA synthesis procedure was performed using the extracted RNA obtained according to Example 4a. The reverse transcription reagent kit facilitated cDNA synthesis of the extracted RNA prior to analysis by qPCR. Reagents from a PRIMESCRIPT RT Reagent Kit (Perfect Real Time, Takara Bio Inc., Catalogue No. RR037B) were obtained. The Kit included 5X PRIMESCRIPT buffer, Random 6mers, and PRIMESCRIPT RT Enzyme Mix I referenced below. The cDNA synthesis procedure was performed according to Steps 1 to 9 described below.

[0316] Step 1. The PRIMESCRIPT RT Reagent Kit was removed from the -20 °C freezer. The 5X PRIMESCRIPT Buffer and Random 6mers were thawed at room temperature. The PRIMESCRIPT RT Enzyme Mix I was kept on ice at about 0 °C.

[0317] Step 2. Once the reagents were thawed, the 5X PRIMESCRIPT Buffer and Random 6mers were vorexed for 5 seconds.

[0318] Step 3. The PRIMESCRIPT RT Enzyme Mix I was mixed by pipetting. The PRIMESCRIPT RT Enzyme Mix I was not vortexed.

[0319] Step 4. The Mastermix was prepared by adding 4 pL of 5X PRIMESCRIPT Buffer, 4 pL of Random 6mers and 1 pL of PRIMESCRIPT RT Enzyme Mix I per sample to a 2 mL tube.

[0320] Step 5. 9 pL of the Mastermix was added to a well of a 96-well PCR plate.

[0321] Step 6. 11 pL of extracted RNA was added to the well of the 96-well PCR plate for a total well volume of 20 pL.

[0322] Step 7. The 96-well PCR Plate was sealed with an aluminum seal and vortexed for 10 seconds, then centrifuged for 1 minute at 3,000 rpm.

[0323] Step 8. The 96-well PCR plate was incubated using a thermocycler under the conditions for the steps provided in Table 7 below.Table 7.

[0324] Step 9. When the thermocycler reached 4 °C, the 96-well PCR plate was removed from the thermocycler and stored at -20 °C until RT-PCR analysis.Example 4c

[0325] Twenty-four whole-urine samples were tested: (a) twelve of the twenty-four urine samples were collected from male human subjects who have a prostate and who did not have a DRE on the same day as and before providing the urine sample (non-same-day-DRE urine); and (b) the other 12 urine samples were collected from male human subjects who have a prostate and who had a DRE within about 180 minutes before providing the urine sample (post-DRE urine). RNA was extracted from the non-same-day-DRE urine and post-DRE urine according to Example 4a or the method described in the Norgen Urine Total RNA Purification Maxi 96-Well Kit (Slurry Format) User Manual (“Norgen method”, available at: https: / / norgenbiotek.com / product / urine-total-rna- purification-maxi-kit-slurry- format?srsltid=AfmBOopVZAEBwz9GmMeOnBOKJD3IlxvWRa_zpFYwfE988aDJrkHZ4Mu3).

[0326] The RNA extracted from non-same-day-DRE and post-DRE urine was reverse transcribed to cDNA according to the method of Example 4b. The average Crt value for the 18 genes of Table 4 was evaluated for the cDNA reverse transcribed from the RNA extracted from the non-same-day- DRE and post-DRE urine samples.

[0327] Using non-same-day-DRE urine, detection of cDNA reverse transcribed from RNA extracted according to the method of Example 4a (94% 18-gene average cDNA detection) was significantly greater than detection of cDNA reverse transcribed from RNA extracted according to the Norgen method (77% 18-gene average cDNA detection) (Table 8). Also using non-same-day-DRE urine, detection of cDNA reverse transcribed from RNA extracted according to the method of Example 4a required significantly fewer Crt cycles (indicating stronger cDNA signals) than for cDNA reverse transcribed from RNA extracted according to the Norgen method (Table 9). Overall, the cDNA reverse transcribed from the RNA extracted from non-same-day-DRE urine according to the method of Example 4a had a 19.59x increase compared to the cDNA reverse transcribed from the RNA extracted according to the Norgen method (Table 9).Table 8. Improvement of detection of cDNA reverse transcribed from mRNA extracted from non- same-day-DRE urine of Example 4aTable 9. Detection of cDNA reverse-transcribed from mRNA extracted from non-same-day-DRE urine of Example 4a

[0328] Using post-DRE urine, detection of cDNA reverse transcribed from RNA extracted according to the method of Example 4a (96% 18-gene average cDNA detection) was greater than detection of cDNA reverse transcribed from RNA extracted according to the Norgen method (95% 18-gene average cDNA detection) (Table 10). Moreover, there was a significant improvement in detection of cDNA reversed transcribed from RNA expressed by ERG and T2ERG using the RNA extraction method of Example 4a compared to the Norgen method (Table 10). Also using post-DRE urine, detection of cDNA reverse transcribed from RNA extracted according to the method of Example 4a required fewer Crt cycles (indicating stronger cDNA signals) than for cDNA reverse transcribed from RNA extracted according to the Norgen method (Table 11). Overall, the cDNA reverse transcribed from the RNA extracted from post-DRE urine according to the method of Example 4a had an 8.77x increase compared to the cDNA reverse transcribed from the RNA extracted according to the Norgen method (Table 11).Table 10. Improvement of detection of cDNA reverse transcribed from RNA extracted from post-DRE urine of Example 4aTable 11. Detection of cDNA reverse-transcribed from RNA extracted from post-DRE urine ofExample 4a

Claims

WHAT IS CLAIMED IS:

1. A method for extracting RNA present in whole urine from a subject’s urine sample, comprising: admixing the whole urine and a first solid support to provide an RNA-bound first solid support, wherein the first solid support comprises silicon carbide; admixing the RNA-bound first solid support and a polar organic solvent to provide an RNA- bound first solid support admixture and performing centrifugation of the RNA-bound first solid support admixture to provide a pellet and a supernatant; removing the supernatant, and washing the pellet with a wash buffer to provide a washed RNA-bound first solid support; admixing wash buffer, the washed RNA-bound first solid support, and a second solid support to provide an RNA-bound second solid support, wherein the second solid support comprises a silicon-based compound; washing the RNA-bound second solid support to provide a washed RNA-bound second solid support; treating the washed RNA-bound second solid support with deoxyribonuclease (DNase) to provide a DNase-treated second solid support and a flowthrough; washing the DNase-treated second solid support to provide a washed DNase-treated second solid support; drying the washed DNase-treated second solid support to provide a dried second solid support; and eluting RNA from the dried second solid support to provide extracted RNA.2 The method of claim 1, wherein the whole urine comprises first-catch urine.3 The method of claim 1 or 2, wherein the whole urine has a volume of about 5 mL to about 10 mL.4 The method of any one of claims 1-3, wherein the whole urine has a volume of about 5 mL.5 The method of any one of claims 1-4, wherein the method does not comprise isolating an exosome from the whole urine.

6. The method of any one of claims 1-5, wherein the first solid support is one of a plurality of silicon carbide particles, and the method comprises admixing the whole urine and the plurality of silicon carbide particles.

7. The method of claim 6, wherein the plurality of silicon carbide particles are present in a slurry, and the method comprises admixing the whole urine and the slurry.8 The method of claim 7, wherein the admixing the whole urine and the slurry occurs at a concentration of about 1 :0.07 (v / v) whole urine:slurry.9 The method of any one of claims 1-8, wherein the admixing the whole urine and the first solid support, plurality of silicon carbide particles, or slurry occurs in the presence of a lysis buffer.10 The method of claim 9, wherein the lysis buffer comprises P-mercaptoethanol.11 The method of claim 10, wherein the P-mercaptoethanol is present in the lysis buffer at a concentration of up to about 5% by volume of the lysis buffer.12 The method of claim 10, wherein the P-mercaptoethanol is present in the lysis buffer at a concentration of about 1% by volume of the lysis buffer.13 The method of any one of claims 9-12, wherein the lysis buffer comprises guanidine or a salt thereof.14 The method of any one of claims 1-13, wherein the polar organic solvent is ethanol.15 The method of any one of claims 1-14, wherein performing the centrifugation of the RNA- bound first solid support admixture is for about 5 minutes at a centrifugal force of about 2,000 x g.16 The method of any one of claims 1-15, wherein performing the centrifugation of the RNA- bound first solid support admixture occurs at a temperature of about 23 °C to about 30 °C.17 The method of any one of claims 1-16, wherein the admixing the wash buffer, the washed RNA-bound first solid support, and the second solid support comprises performing a centrifugation of the wash buffer, washed RNA-bound first solid support, and the second solid support to provide the RNA-bound second solid support.18 The method of any one of claims 1-17, wherein the silicon-based compound is silica and the second solid support is one of a plurality of silica particles.19 The method of any one of claims 1-17, wherein the silicon-based compound is silicon carbide, and the second solid support is one of a plurality of silicon carbide particles.

20. The method of claim 18 or 19, wherein the silica particles or silicon carbide particles are present in a slurry.

21. The method of claim 18 or 19, wherein the silica particles and silicon carbide particles are present in a slurry.

22. The method of claim 18 or 19, wherein the silica particles or silicon carbide particles are contained in a column.

23. The method of claim 18 or 19, wherein the silica particles and silicon carbide particles are contained in a column.

24. The method of any one of claims 17-23, wherein performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, and the second solid support is at a centrifugal force of about 3,000 x g.

25. The method of any one of claims 17-24, wherein performing the centrifugation of the wash buffer, the washed RNA-bound first solid support, and the second solid support occurs at a temperature of about 23 °C to about 30 °C.

26. The method of any one of claims 1-25, wherein the washing the RNA-bound second solid support comprises admixing the RNA-bound second solid support and a wash buffer to provide an RNA-bound second solid support and wash buffer admixture, and performing a centrifugation of the RNA-bound second solid support and wash buffer admixture to provide the washed RNA-bound second solid support.

27. The method of claim 26, wherein the wash buffer has a volume of about 400 pL.

28. The method of claim 26 or 27, wherein performing the centrifugation of the RNA-bound second solid support and wash buffer admixture is at a centrifugal force of about 3,000 x g.

29. The method of any one of claims 26-28, wherein performing the centrifugation of the RNA- bound second solid support and wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C.

30. The method of any one of claims 1-29, wherein the treating comprises performing a centrifugation of the washed RNA-bound second solid support and deoxyribonuclease (DNase) to provide the DNase-treated second solid support and the flowthrough.

31. The method of claim 30, wherein the DNase is DNase I.

32. The method of claim 30 or 31, wherein performing the centrifugation of the washed RNA- bound second solid support and the DNase is at a centrifugal force of about 3,000 x g.

33. The method of any one of claims 30-32, wherein performing the centrifugation of the washed RNA-bound second solid support and the DNase occurs at a temperature of about 23 °C to about 30 °C.

34. The method of any one of claims 30-33, wherein the flowthrough is a first flowthrough, wherein the treating further comprises admixing the DNase-treated second solid support and the first flowthrough to provide a DNase-treated second solid support admixture and incubating the DNase- treated second solid support admixture at room temperature for about 15 minutes to provide an incubated DNase-treated solid support admixture.

35. The method of claim 34, further comprising admixing the incubated DNase-treated second solid support admixture and a wash buffer to provide a DNase-treated second solid support and wash buffer admixture and performing a centrifugation of the DNase-treated second solid support and wash buffer admixture to provide the washed DNase-treated second solid support and a second flowthrough.

36. The method of claim 35, wherein performing the centrifugation of the DNase-treated second solid support and wash buffer admixture is at a centrifugal force of about 3,000 x g.

37. The method of claim 35 or 36, wherein performing the centrifugation of the DNase-treated second solid support and wash buffer admixture occurs at a temperature of about 23 °C to about 30 °C.

38. The method of any one of claims 35-37, further comprising discarding the second flow through after performing the centrifugation of the DNase-treated second solid support and wash buffer admixture.

39. The method of any one of claims 35-38, further comprising repeating the steps of admixing the DNase-treated second solid support and the wash buffer and performing a centrifugation to provide the washed DNase-treated second solid support.

40. The method of any one of claims 1-39, wherein the drying comprises performing a centrifugation of the washed DNase-treated solid support at a centrifugal force of about 3,000 x g for up to about 20 minutes.

41. The method of claim 40, wherein the performing the centrifugation of the washed DNase- treated second solid support is for about 5 minutes to about 15 minutes.

42. The method of claim 40, wherein the performing the centrifugation of the washed DNAase- treated second solid support is for about 15 minutes.

43. The method of any one of claims 40-42, wherein the performing the centrifugation of the washed DNAase-treated second solid support occurs at a temperature of about 23 °C to about 30 °C.

44. The method of any one of claims 1-43, wherein the eluting comprises admixing the dried second solid support and an elution buffer to provide an elution support admixture, incubating the elution support admixture to provide an incubated elution support admixture, and performing a centrifugation of the incubated elution support admixture to provide the extracted RNA.

45. The method of claim 44, wherein the incubating the elution support admixture is for about 2 minutes.

46. The method of claim 44 or 45, wherein the elution buffer has a temperature of from about 20 °C to about 37 °C.

47. The method of any one of claims 44-46, wherein the elution buffer has a pH of from about 7 to about 8.5.

48. The method of any one of claims 44-47, wherein performing the centrifugation of the incubated elution support admixture is at a centrifugal force of about 500 rpm for about 2 minutes.

49. The method of any one of claims 44-48, wherein performing the centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C to about 30 °C.

50. The method of any one of claims 44-49, wherein the centrifugation of the incubated elution support admixture is a first centrifugation of the incubated elution support admixture, and the method further comprises performing a second centrifugation of the incubated elution support admixture.

51. The method of claim 50, wherein performing the second centrifugation of the incubated elution support admixture is at a centrifugal force of about 3,000 x g for about 2 minutes.

52. The method of claim 50 or 51, wherein performing the second centrifugation of the incubated elution support admixture occurs at a temperature of about 23 °C to about 30 °C.

53. The method of any one of claims 1-52, further comprising detecting the extracted RNA.

54. The method of claim 53, wherein the detecting comprises reverse transcribing the extracted RNA to complementary DNA (cDNA) using a reverse transcriptase and detecting the cDNA.

55. The method of claim 53 or 54, wherein detecting the extracted RNA or the cDNA comprises performing: i) reverse transcription-quantitative polymerase chain reaction (RT-qPCR); ii) RNA sequencing; iii) digital PCR (dPCR); iv) microarray analysis; v) Northern Blot; vi) UV spectroscopy; or vii) fluorometry.

56. The method of claim 54, wherein the detecting is detecting the cDNA, and the detecting the cDNA comprises performing qPCR.

57. The method of any one of claims 54-56, wherein the reverse transcriptase is Moloney murine leukemia virus (M-MLV) reverse transcriptase.

58. The method of any one of claims 1-57, wherein the extracted RNA is mRNA, miRNA, long non-coding RNA, or circular RNA.

59. The method of any one of claims 1-58, wherein the subject is prostate-biopsy naive.

60. The method of any one of claims 1-58, wherein the subject is prostate biopsy-prior negative.

61. The method of any one of claims 1-60, wherein the RNA is expressed by a gene of the subject.

62. The method of claim 61, wherein the gene is a cancer gene.

63. The method of claim 61 or 62, wherein the gene is a bladder cancer gene, a kidney cancer gene, or a prostate cancer gene.

64. The method of any one of claims 61-63, wherein the gene is a prostate cancer gene.

65. The method of claim 63 or 64, wherein the prostate cancer gene is KLK3.

66. The method of claim 63 or 64, wherein the prostate cancer gene is ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRJN3A, LINC00993, LRRN1, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PRAIA, PLA2G7, PRCAT47, SPINK1, TDO2, TK1, 1MEFF2, TRGV9.1, VSTM2L, 1MPRSS2-ERG, SCHLAP1, OR51E2, APOCR PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SPON2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, H0XC6, o KLK3.

67. The method of claim 63 or 64, wherein the prostate cancer gene is ACSM1, AMACR, AR, COL9A2, CRISP 3, CST2, DLX1, ETV1, F5, GDF15, GLYATL1, G0LM1, GRJN3A, LINC00993,LRRNI, MIPEP, MS4A8, MY06, PCA3.1, PDLIM5, PEX10, PI A l A, PLA2G7, PRCAT47, SPINK1, 1'1)02, TK1, TMEFF2, TRGV9.1, or VSIM2L.

68. The method of claim 63 or 64, wherein the prostate cancer gene is TMPRSS2-ERG, SCHLAP1, OR51E2, AP0C1, PCAT14, CAMKK2, PCA3, NKAIN1, B3GNT6, TFF3, SP0N2, PCGEM1, TRGV9, TMSB15A, ERG, KLK4, H0XC6, or KLK3.

69. The method of claim 63, wherein the gene is a bladder cancer gene.

70. The method of claim 63 or 69, wherein the bladder cancer gene is BMPR1B, CDH2, COL7A1, FGFR2, HDHD3, ICAM1, JCAD, PFKFB4, MIR15A, MIR150, EGFL7, MIR210, MIRLET7A1, MIRLET7A2, MIRLET7A3, MIRLET7B, MIRLET7C, MIRLET7D, MIRLET7G, MIR30C1, MIR200A, MIR34A, MIR365A, MIR205, MIR497, EGLN3, and SOD2.

71. The method of claim 63, wherein the gene is a kidney cancer gene.

72. The method of claim 63 or 71, wherein the kidney cancer gene is ABL1, ANXA10, UPK1B, CRH, IGF2, H0XA13, MDK, CXCR2, IGFBP5, ROBO1, WNT5A, CDC42BPB, KIFC3, KRT20, LCN2, MAGEA3, RPS21, SLC1A6, XIAP, CDK1, PRSSI, CASPI4, KLHDC7B, ANRTL, MKLN1-AS, TALAM1, TTN-AS1, UCA1, LNMAT2, BCYRN1, GAS5, CD24, TOP2A, IQGAP3, UBE2C, IGFBP5, BIRC5, MIR29B2, MIR31, MIR141, MIR34B, MIR10B, MIR103A1, MIR103A2, MIR103B1, MIR145, MIR182, MIR205HG, MIR20A, MIR92A2, MIR17, MALAT1, PCAT-1, SPRY4-IT1, HOTAIR, HYMAI, CCDC148-AS1, CCDC86-AS1, KDM4C, ITCH, ACVR2A, MTDH, ATIC, TAF4B, RBM3, or GLI3.

73. The method of any one of claims 1-72, wherein the extracted RNA is provided in an amount that is at least 3-fold greater than extracted RNA obtained from a method that does not comprise admixing whole urine and a first solid support comprising silicon carbide.

74. The method of any one of claims 1-73, wherein the urine sample is provided outside a clinical setting.

75. The method of any one of claims 1-74, wherein the subject is a human subject.

76. The method of any one of claims 1-75, wherein the subject is a male human subject.

77. The method of claim 76, wherein the male human subject has a prostate.

78. The method of any one of claims 1-77, wherein the subject has or is suspected of having prostate cancer.

79. The method of claim 78, wherein the prostate cancer is Grade Group (GG) >2 prostate cancer.

80. The method of claim 78 or 79, wherein the prostate cancer is GG >3 prostate cancer.

81. The method of any of claims 78-80, wherein the prostate cancer is GG >4 prostate cancer.

82. The method of any one of claims 78-81, wherein the prostate cancer is GG5 prostate cancer.

83. A kit comprising a container for containing the urine sample of any one of claims 1-82.

84. The kit of claim 83, further comprising instructions for providing the urine sample.

85. The kit of claim 83 or 84, wherein the container is a sealable container.

86. The kit of claim 85, wherein the sealable container is a stoppered vial.

87. The kit of any one of claims 83-86, wherein the container contains a preservative.

88. The method of any one of claims 1-82, wherein the method does not comprise admixing the whole urine and the first solid support under reduced pressure.

89. The method of any one of claims 1-82 and 88, wherein the method does not comprise admixing the RNA-bound first solid support and ethanol under reduced pressure.

90. The method of any one of claims 1-82, 88 and 89, wherein the method does not comprise admixing the wash buffer, the washed RNA-bound first solid support and the second solid support under reduced pressure.

91. The method of any one of claims 88-90, wherein the reduced pressure is about 0.8 bar.

92. A composition comprising whole urine and a solid support comprising silicon carbide.

93. The composition of claim 92, wherein the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

94. The composition of claim 92 or 93, wherein the solid support is one of a plurality of solid supports, and the composition comprises the plurality of solid supports.

95. A composition comprising whole urine and an RNA-bound solid support.

96. The composition of claim 95, wherein the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

97. The composition of claim 95 or 96, wherein the RNA-bound support is one of a plurality of RNA-bound solid supports, and the composition comprises the plurality of RNA-bound supports.

98. The composition of any one of claims 95-97, made by a method comprising admixing whole urine and a solid support comprising silicon carbide to provide the RNA-bound solid support, wherein the admixing is not performed under reduced pressure.

99. The composition of any one of claims 95-97, made by a method comprising admixing whole urine and a plurality of solid supports comprising silicon carbide to provide the plurality of RNA- bound solid supports, wherein the admixing is not performed under reduced pressure.

100. The composition of claim 98 or 99, wherein the reduced pressure is about 0.8 bar.

101. A method for making an RNA-bound solid support, comprising admixing whole urine and a solid support comprising silicon carbide to provide the RNA-bound solid support.

102. The method of claim 101, wherein the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

103. The method of claim 101 or 102, wherein the method does not comprise admixing the whole urine and the solid support under reduced pressure.

104. A method for making a plurality of RNA-bound solid supports, comprising admixing whole urine and a plurality of solid supports comprising silicon carbide to provide the plurality of RNA- bound solid supports.

105. The method of claim 104, wherein the whole urine is from a urine sample of a male human subject who has a prostate and who did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

106. The method of claim 104 or 105, wherein the method does not comprise admixing whole urine and the plurality of solid supports under reduced pressure.

107. The method of claim 106, wherein the reduced pressure is about 0.8 bar.

108. The method of any one of claims 1-82 and 88-91, wherein the subject did not have a digital rectal examination (DRE) within about 180 minutes before providing the urine sample.

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