Compositions and methods for isolating eukaryotic mRNA from stool samples

Abstract

The present disclosure provides methods and compositions, e.g., kits, for isolating eukaryotic nucleic acid from a stool sample. In one embodiment, the method provided herein involves incubating the stool sample with a chaotropic salt, heating the stool sample, and using a substrate coupled to a locked nucleic acid (LNA) oligonucleotide to isolate the nucleic acids hybridized to the LNA oligonucleotide.

Description

COMPOSITIONS AND METHODS FOR ISOLATING EUKARYOTIC MRNA FROM STOOL SAMPLES SEQUENCE LISTING

[0001] The sequence listing that is contained in the file named “088177-8005WO01”, which is 106,133 bytes and was created on September 09, 2025, is filed herewith by electronic submission and is incorporated by reference herein. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to US provisional application 63 / 692,691, filed September 9, 2024, the disclosure of which is incorporated herein by reference. FIELD OF THE INVENTION

[0003] The present disclosure generally relates to molecular biology and diagnostics. In particular, the present disclosure relates to compositions and methods for the separation or isolation of nucleic acid(s) from stool samples for the purpose of research, diagnostic screening, disease prognosis, or therapeutic benefit prediction. BACKGROUND

[0004] The ability to isolate and analyze human RNA from various samples or products, e.g., body fluids or tissues, is important for genetic and genomic testing, cancer screening, analysis of treatment benefit, analysis of disease progression, and other diagnostic and research areas (Blandino et al., 2023; Byron et al., 2016). The ability to measure the amount of various specific human RNA or DNA in stool derived from exfoliated cells or vesicles present in human stool is a promising approach to noninvasively sample the digestive tract or gut. In addition to being noninvasive, stool has the strong advantage that, unlike tissue-based colon transcriptomics, it samples the entire colon. Indeed, recent research has indicated that stool- based transcriptomics provides superior information regarding the status of the gut health compared to colon tissue biopsies (Dan et al., 2023; Ungar et al., 2022).

[0005] Efficient enrichment for human RNA and DNA during isolation of host from stool samples is difficult, unknown, and unpredictable as most commercial kits that are designed for nucleic acid isolation from stool are optimized for purification of bacterial nucleic acids rather than host eukaryotic nucleic acids. The ability to isolate both host RNA and host DNA simultaneously, directly from crude stool samples has not been demonstrated previously. Stool is particularly challenging when compared to tissue, because the variety of ingested and undigested foods, large amounts of microbial background nucleic acid, the presence of bile, bilirubin, hemoglobin, mucus and other body waste products complicates the samplecomposition and human mRNA and nucleic acid extraction and quantification (Reck et al., 2015).

[0006] In tissues, human mRNA represents a small fraction of the total RNA present: ~5% of RNA in a cell is mRNA, the rest being mostly ribosomal / tRNA and mitochondrial RNA. In stool, the fraction of human mRNA compared to total RNA is further diluted by the large amount of microbial RNA that is present in fecal samples. For techniques that perform better using pure human mRNA input, such as RNA-seq or some diagnostic techniques (Zhao et al., 2018), isolation and purification of human mRNA is a necessary step. Most techniques for the isolation of eukaryotic (including human) mRNA require a two-step complete nucleic acid extraction from crude lysate prior to mRNA isolation.

[0007] Nearly all eukaryotes modify their mRNA at the time of synthesis by adding a long polyA tail to the 3’ end of mature mRNA molecules. By selecting for the presence of these polyA tails, the eukaryotic mRNA fraction of RNA can be isolated and purified from total RNA by a method called polydT capture. PolydT capture typically follows total RNA extraction in a two-step procedure commonly used to enrich for eukaryotic mRNA. Unfortunately, the two- step approach that selects for mRNA following total RNA extraction is time consuming, expensive, and the multiple handling steps can result in poor nucleic acid(s) recovery due to loss from additional sample processing. The use of locked nucleic acids (LNA) for the direct selection of mRNA from tissue has been previously shown (Jacobsen et al., 2004, PCT / IB2003 / 006354). However, the application of these chemically modified locked nucleic acids (LNA) has not previously been conceived or demonstrated in what is perhaps the most difficult medium, stool, so functionality was unknown, and utility was unpredictable. In addition, Jacobsen et al didn’t envision the targeted isolation of various RNA molecules (such as long noncoding RNA, miRNA), fragmented mRNA molecules, nor the co- isolation of the DNA and RNA molecules at the same time on single surfaces.

[0008] Stool is a potentially rich source of diagnostic, prognostic, and treatment predictive information. In particular, much attention has been paid to the gut microbiome, colorectal cancer screening and infectious disease detection using stool (Hou et al., 2022; Kasirga et al., 2019). Unfortunately, the use of host nucleic acid present in stool as a potential source of diagnostic and prognostic information has been neglected (He et al., 2019) due to sample complexity and functional difficulties.

[0009] The majority of commercial products to extract nucleic acid from stool are designed to extract microbiome derived nucleic acid. To our knowledge, there is no dedicatedcommercial product to extract pure human host nucleic acids from fecal material. To some extent this can be explained by the difficulty in extracting, enriching and purifying host nucleic acid from stool. The majority of nucleic acid in stool is microbiome derived. Less than 1% of the dry weight of stool consists of host cells containing host nucleic acids while 12% consists of bacterial cells containing bacterial nucleic acids (Ryan et al. 2021). In addition, stool is a very diverse mixture of undigested debris, a multitude of PCR inhibitors, mucus, bacterial proteins and digestive enzymes (Ryan et al. 2021). This greatly complicates extracting the small amount of host nucleic acid that is present in a form suitable for downstream applications such as PCR or next generation sequencing, methods that perform best with high quality and high purity nucleic acid. Extracting nucleic acid from stool is not the same as nucleic acid extraction from blood, or other tissues in which host cells and nucleic acid are abundant.

[0010] Previous investigations (PCT / IB2003 / 006354) have focused on the use of LNA technology to capture polyadenylated (polyA) eukaryotic mRNA from human tissue or fluids. Use of LNA technology to capture polyadenylated (polyA), non-polyadenylated eukaryotic RNA, eukaryotic DNA, or simultaneously capturing eukaryotic RNA and DNA from stool was unknown and unpredictable given the low abundance of target nucleic acids, and the semi-solid form and complexity of the sample. The previous investigations do not address the negative impact of PCR inhibitors on analytical detection of nucleic acid molecules. The presence of PCR inhibitor’s impact on extraction of nucleic acid from stool was previously unknown and determined to be highly variable with subject. Here we demonstrate LNA capture technology which has been reconceived and successfully developed to perform polyA selection and capture of specific nucleic acid (RNA or DNA) target sequences in stool simultaneously. Although the procedure uses LNAs in the methodology, the procedure required non-obvious modification without which the procedure would not work to capture both intact and fragmented nucleic acid molecules, simultaneously or separately, in stool samples hence is not the same methodology as that used for other tissue types.

[0011] Isolating eukaryotic nucleic acid(s) directly from crude lysate in a one-step procedure can result in considerable cost and time savings. Indeed, there are a number of commercial products on the market that claim to be able to directly capture eukaryotic mRNA from crude cell or tissue lysates using polydT capture (e.g., “DynabeadsTMmRNA DIRECTTMPurification Kit,”). Unfortunately, none of the existing solutions have been engineered to work in stool or other complex body fluids which are particularly challenging matrices. Our invention is designed to fill this gap and provide a robust solution for the direct one stepisolation of both intact and fragmented simultaneously from human RNA of stool samples as well as other challenging matrices.

[0012] Given these challenges, a robust extraction method that is sensitive enough to detect disease-related host nucleic acid must have the ability to disaggregate and dissolve the stool and to; process and concentrate host nucleic acid(s) from a large volume of stool; remove the inhibitors concentrated within the sample; robustly lyse host cells and extracellular vesicles embedded in stool, run on automated platforms, and easily scale up or down to process various stool volumes. Commercial kits and phenol chloroform based approaches can robustly extract total nucleic acids from smaller amounts of stool. However, these methods mostly isolate total nucleic acids so no enrichment of host nucleic acids is performed. This limits the amount of host nucleic acid that can be input into molecular diagnostics detection techniques such as qPCR or RT-qPCR.

[0013] There are published methods to enrich the human signal present in stool. One method for isolation of host cells from stool is differential centrifugation. This is the extraction technique used by the recently FDA-approved multi-target RNA test14. After initial processing, stool samples are subject to differential centrifugation in an aqueous buffer such that the host cells are pelleted while the smaller bacterial cells are left in solution. This process is repeated several times until a pellet, enriched for host cells, is obtained. This pellet is then extracted using commercial extraction methods.

[0014] While this method can highly enrich some host cells from a stool sample, repeated centrifugations are cumbersome, time consuming, difficult to automate, yield low amounts of mRNA and only isolate mRNA from a fraction of the fully intact cells that are present in the stool sample. Cells trapped in the stool matrix, attached to cellular debris or stool microparticles will be lost in the differential centrifugation process. Also the RNA from partially lysed cells with intact nuclei, and RNA in extracellular vesicles will be lost because they will move differently in the centrifugal field than intact cells, making the final yield of host RNA low, and perhaps not representative of all the host cells actually present in stool.

[0015] Another possibility is to use magnetic beads coated with anti-EPCAM antibodies to attach to and isolate colonic cells from stool15,16. While this method is simpler than differential centrifugation it would also miss any host cells present which don’t express EPCAM cell surface marker, potentially missing a large part of the host RNA signal in stool, for example signal from immune cells.SUMMARY OF INVENTION

[0016] The present disclosure in one aspect provides a method of isolating eukaryotic nucleic acid from a crude biological sample, e.g., a stool sample.

[0017] In one embodiment, the method comprises a) obtaining a stool sample from a subject, wherein the stool sample comprises at least an nucleic acid from the subject; b) incubating the stool sample with a solution containing a chaotropic salt; c) heating the stool sample to a temperature between 60°C and 90°C; d) incubating the stool sample with a substrate coupled to a locked nucleic acid (LNA) oligonucleotide, wherein the LNA oligonucleotide comprises a sequence complementary to the nucleic acid(s)A, thereby allowing the nucleic acid(s) to hybridize to the LNA oligonucleotide; and e) isolating the nucleic acid(s) hybridized to the LNA oligonucleotide.

[0018] In some embodiments, the chaotropic salt is guanidinium isothiocyanate (GITC) or guanidinium thiocyanate (GTC). In some embodiments, the stool sample is incubated with the chaotropic salt at a concentration of about 1.5M in step (b). In some embodiments, the solution containing the chaotropic salt is a preservation buffer or lysis buffer. In some embodiments, the stool sample is heated for 1 to 15 min minutes in step c).

[0019] In some embodiments, the LNA oligonucleotide comprises poly-deoxythymidine (polydT). In some embodiments, the LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs: 1-9.

[0020] In some embodiments, the nucleic acid is a DNA or RNA, e.g., mRNA, long non- coding RNA, microRNA.

[0021] In some embodiments, the substrate is couple to a second LNA oligonucleotide comprising a sequence complementary to an internal sequence of the nucleic acid. In some embodiments, the second LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs: 10-25.

[0022] In some embodiments, the subject is a human.

[0023] In some embodiments, the substrate is a magnetic, a paramagnetic, or a super paramagnetic bead.

[0024] In some embodiments, the oligonucleotide is coupled to the substrate via a biotin modified 5’ end. In some embodiments, the oligonucleotide is coupled to the substrate via a 5’ amine modified end with a carbon spacer or other standard coupling scheme used to attach nucleic acids to solid surfaces.

[0025] In some embodiments, the method further comprises detecting a biomarker in the isolated nucleic acid(s).

[0026] In some embodiments, the oligonucleotide is coupled to the substrate via a biotin modified 5’ end, a carboxyl amine coupling or any one of the usual ways of coupling nucleic acids to surfaces. In some embodiments, the oligonucleotide is coupled to the substrate via a 5’ amine modified end with a carbon spacer.

[0027] In some embodiments, the method is executed via an automation.

[0028] In another aspect, the present disclosure provides a kit for isolating nucleic acid(s) from a stool sample. In some embodiments, the kit comprises: reagents for making a solution comprising a chaotropic salt; and reagents for making a substrate coupled to a LNA oligonucleotide comprising a sequence complementary to the nucleic acid(s) to be isolated. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which are incorporated herein, form part of the specification. Together with this written description, the drawings further serve to explain the principles of, and to enable a person skilled in the relevant art(s), to make and use the present invention.

[0030] FIG.1 illustrates the results of an exemplary method of isolating mRNA from stool samples using LNA-coupled magnetic beads.

[0031] FIG.2 illustrates the comparison of the results of an exemplary method of isolating mRNA disclosed herein with the methods of using a commercial kit. Simply replacing standard capture probes with LNA containing probes did not result in improved isolation compared to the commercial kits. TFS refers to the DynabeadsTMmRNA DIRECTTMPurification Kit.

[0032] FIG. 3 illustrates that the combination of using GITC and heating improves the mRNA isolation from human stool samples using LNA beads. In contrast, addition of GITC without heating interferes with the isolation of mRNA using a commercially available stool sample mRNA isolation kit. TFS refers to the DynabeadsTMmRNA DIRECTTMPurification Kit.

[0033] FIG. 4 illustrates that the combination of using GITC and heating improves the mRNA isolation from human stool samples using LNA beads across 6 different stool samples to prove this is a general requirement for more effective mRNA extraction.

[0034] FIG.5 illustrates that capture of mRNA by using LNA oligonucleotides targeting internal sequence of the mRNA resulted in higher yields (i.e., lower Cts) than using LNA oligonucleotides comprising poly dT when the RNA is fragmented. In samples with intact mRNA polydT had higher yield, while in samples with fragmented mRNA target capture isolated the most mRNA.

[0035] FIG.6A-6C illustrates that increased stool input in an exemplary extraction process described herein improves PCR Cts.

[0036] FIG. 7A-7G illustrates that an exemplary extraction process described herein removed more PCR inhibitors from stool extractions than commercial stool extraction kits. FIG. 7A: The extraction processing schematic. FIG. 7B and 7C: Cts for 2 mRNA targets extracted by Phenol method, Silica beads or ECB Extract. FIG. 7D-7F. A 0.5 gram stool extraction was run for 6 clinical samples using each extraction method. After extraction increasing amounts of eluate were added to RT-qPCR reactions and the resulting Cts measured by Taqman assay using our mRNA-specific Taqman assay for GAPDH. FIG. 7G: The same samples were extracted by either the Phenol method or ECB Extract then MS2 phage was spiked into the extracts and the PCR Cts measured and compared to MS2 spiked into water. The difference in Ct between water and extract is shown. Several of the Omega eluates failed to amplify, those were assigned a Ct of 40 for making this graph.

[0037] FIG.8A-8C shows the GAPDH 3’5’ assay Delta Ct. We classified assays with a < 1 Ct difference in Ct as slightly to moderately degraded, and assays with a > 1Ct difference as severely degraded. We extracted those samples with the 3 extraction methods (poly dT, target capture only, ECB extract (both poly dT + target capture) and found that ECB extract consistently gave the best Cts for either of 2 mRNAs, GAPDH and TGFB1.

[0038] FIG.9A-9F shows the clinical performance comparison between ECB Extract and Phenol method. FIG. 9A: Plotted for each gene is CRC AUC comparing Phenol method (Y- axis) with ECB Extract (X-axis). Genes below the diagonal represent genes with better CRC diagnostic performance using ECB Extract compared to Phenol method. FIG.9B and 9C: CRC diagnostic ROC curves for each gene are plotted for Phenol method and ECB Extract. FIG. 9D: Plotted for each gene is APL AUC comparing Phenol method (Y-axis) with ECB Extract (X-axis). Genes below the diagonal represent genes with better APLA diagnostic performance using ECB Extract compared to Phenol method. FIG.9E and 9F: Plotted for each gene is the APLA diagnostic ROC curves for Phenol method and ECB Extract.

[0039] DETAILED DESCRIPTION OF THE INVENTION

[0040] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0043] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0044] Definitions

[0045] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

[0046] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0047] As used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or”, unless the context clearly dictates otherwise. The term “based on” is not exclusive and allowed for being based on additional factors not described unless the context clearly dictates otherwise. In addition, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise. The meaning of “in . . .” includes “within ...” and “on ...”.

[0048] The terms “assessing”, “assaying”, “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “measuring a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.

[0049] As used herein, the term “biomarker” refers to a detectable organic biomolecule associated with a particular phenotype or risk of developing a particular phenotype, such as a polynucleotide (e.g., RNA (e.g., mRNA) or DNA (e.g., cDNA)) or a polypeptide, which is differentially present in a biological sample taken from a subject having a certain clinically aberrant condition (e.g., having a gastrointestinal disease or a high-risk feature associated with progression to gastrointestinal disease) as compared to a comparable biological sample taken from a subject who does not have such condition, such as a healthy subject or a non-cancer patient. For example, a biomarker can be a polynucleotide, such as RNA (e.g., mRNA), which is present at an elevated or decreased level in a biological sample (e.g., a tissue sample, a feces sample, or a blood sample) of a gastrointestinal disease patient compared to a comparable sample (e.g., a feces sample) of a subject with a negative diagnosis (e.g., a healthy subject).

[0050] The term “colon cancer” used interchangeably with the term “colorectal cancer” or “rectal cancer” refers to any cancerous neoplasia of the colon (including the rectum and appendix). Many colorectal cancers arise from precancerous colorectal adenomas or adenomatous polyps or advanced precancerous lesions, which are usually benign, but some may develop into cancer over time. The diagnosis of localized colon cancer is often through colonoscopy. Once localized colon cancer is diagnosed, it is usually surgically removed and then may be treated with chemotherapy. Precancerous colorectal adenomas or colorectal adenomatous polyps are a risk factor for colorectal cancer. The removal of colorectal adenomatous polyps at the time of colonoscopy would reduce the risk of having colorectal cancer. In addition, clinical data has shown that early detection and curative surgical resection of colorectal cancer will significantly improve survival rates.

[0051] It is noted that in this disclosure, terms such as “comprises”, “comprised”, “comprising”, “contains”, “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of” and “consists essentially of” have the meaning attributed in United States Patent law; they allow for the inclusion ofadditional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The terms “consists of” and “consisting of” have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.

[0052] As used herein, the term “complementary” and “complementarity” refer to nucleotides (e.g., a nucleotide) or a polynucleotide (e.g., a sequence of nucleotides) associated with base pairing rule. For Example, sequence 5’-A-G-T-3’ is complementary to sequence 3’- T-C-A-5’. Complementary can be “partial,” in which only some nucleic acid bases are matched according to the base pairing rule. Alternatively, there may be “completely” or “total” complementarity between nucleic acids. The complementary degree between nucleic acid chains affects the efficiency and strength of hybridization between nucleic acid chains. This is especially important in the amplification reactions and detection methods that depend upon binding between nucleic acids.

[0053] The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high- performance liquid chromatography.

[0054] As used herein, the term “Locked Nucleic Acid” or “LNA” refers to a class of nucleic acid analogs in which the ribose ring of the nucleotide is chemically modified by the introduction of a methylene bridge connecting the 2'-oxygen and the 4'-carbon. This bridge "locks" the ribose in the 3'-endo conformation, which is the preferred conformation of natural RNA. The structural rigidity conferred by this locking mechanism enhances the hybridization properties of LNA oligomers when bound to complementary DNA or RNA sequences.

[0055] As used herein, the term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, microRNA, cDNA, shRNA, single- stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0056] As used herein, the term “polymerase chain reaction” or “PCR” is used to refer to a technique for amplifying a target sequence and is well recognized by one skilled in the art. The PCR generally consists of introducing a large excess of two oligonucleotide primers into the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. For amplification, the mixture is denatured and then the primers annealed with its complementary sequence within the target molecule. After annealing, the primers were extended with a polymerase so as to form a new pair of complementary chains. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (that is, denaturation, annealing, and extension constitute one “cycle” and there can be numerous “cycles”) to obtain a high concentration of amplified fragments of the desired target sequence. The length of the amplified fragment of the target sequence is determined by the relative position of the primers with respect to each other, so the length is a controllable parameter. Since the desired amplified fragment of the target sequence becomes the predominant sequence (in terms of concentration) in the mixture, it is called “PCR amplified”, “PCR products” or “amplicons”.

[0057] The term “nucleic acid detection assay” refers to any method of determining the nucleotide composition of the target nucleic acid. Nucleic acid detection assay includes but is not limited to DNA sequencing methods and nucleic acid hybridization methods.

[0058] The term “PCR inhibitor” refers to any molecule that interferes with either the reverse transcriptase, polymerase, or equivalent enzymes or processes such that the resultant analytical assessment is not representative of the actual number of molecules in the sample.

[0059] The term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digestion or is produced synthetically, that is capable of acting as a point of initiation of synthesis when placed in the conditions for induction of synthesis of product extended from primers complementary to nucleic acid chain (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and under proper temperature and pH). The primer is typically single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. In the case of double chains, the primer is first treated to separate its strands before being used to prepare the extension product. In general, the primer is an oligo (molecule) of deoxyribonucleotides. At minimum, the length of primer should be sufficient to initiate the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, source of the primer, and the use of the method.

[0060] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient”. A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0061] Method of Purification

[0062] The present disclosure in one aspect provides a method of isolating eukaryotic mRNA from a crude biological sample. In some embodiments, the method described herein directly isolates nucleic acid, for example a nucleic acid of the following types separately or simultaneously; DNA or RNA target molecule including mRNA, long non-coding RNA, microRNA or other information containing nucleic acid molecule, from crude stool or other tissue / fluids lysate using magnetic beads coupled to and covalently bonded to a polydT (poly- deoxythymidine), or other oligonucleotide that is complementary to the nucleic acid, containing locked nucleic acids (LNAs), either directly or with a carbon spacer, by any of the numerous chemistries available to attach an oligonucleotide to a surface.

[0063] LNAs have been demonstrated to increase hybridization specificity and improve PCR assay sensitivity by providing better mismatch discrimination (Ballantyne et al., 2008). Our invention allows us to efficiently use magnetic beads, or other surfaces, to which LNA- containing oligonucleotides have been attached, to capture eukaryotic DNA or RNA target molecules including mRNA, long non-coding RNA, microRNA or other information containing nucleic acid molecule directly out of a crude stool lysate suspended in DNA RNA Shield lysis / preservation buffer, and lysis / preservation buffer to which guanidinium isothiocyanate (GITC) or guanidinium thiocyanate (GTC) has been added.

[0064] We show that the isolation of nucleic acid through its polyA tail (for intact mRNA) or a specific sequence within the nucleic acid (other forms of RNA, DNA or RNA and A simultaneously) is improved by our method. Therefore, in one embodiment, the method disclosed herein comprises a) obtaining a stool or other difficult to lyse or inhibitor laden sample from a biological subject, wherein the sample comprises at least a nucleic acid from the biological subject; b) incubating the sample with a solution containing chaotropic salt; c) heating the sample; d) incubating the sample with a substrate coupled to an oligonucleotide comprising at least 2 or more locked nucleic acid nucleotides (LNA), wherein the oligonucleotide comprises a sequence complementary to the nucleic acid(s) sequence, therebyallowing the nucleic acid(s) to hybridize to the oligonucleotide; and e) isolating the nucleic acid(s) hybridized to the LNA oligonucleotide. In some embodiments, the chaotropic salt is guanidinium isothiocyanate (GITC) or guanidinium thiocyanate (GTC). In certain embodiments, the sample is heated to a temperature between 60 and 90°C for between 1 and 30 minutes in step c). In certain embodiments the sample is clarified by centrifugation, filtration or any of the common methods known in the art for removal of cellular or sample debris after the heating step.

[0065] Oligonucleotides containing LNAs can be purchased from commercial vendors, e.g., IDT, or Qiagen. Preferably, the capture oligonucleotides comprise a double biotin 5’ end, or a 5’ amine modified end with a carbon spacer, followed by a short leader sequence of deoxythymidine (dT). The LNA modified bases can be inserted at approximately every other nucleotide for a total of 8 or 9 LNAs, some examples for one specific case are shown in Table 1 below, wherein symbol “+” represents that the previous nucleotide is a LNA.

[0066] Table 1. Exemplary oligonucleotides containing LNAs some of which contain carbon spacers

[0067] The LNA-containing oligonucleotides can be coupled to a substrate, e.g., a bead, via various methods known in the art. For example, coupling of the biotin LNA-containing oligonucleotides to Dynabeads™ MyOne™ Streptavidin C1 beads can be done as per manufacturer instructions. Streptavidin type beads purchased from other manufactures may provide the similar performance. The stock bottle of mag beads is vortexed for 60 seconds to make sure beads are completely resuspended.0.5 mL mag beads slurry is transferred into a 2 mL low-bind tube. Place on a magnetic rack and pellet beads for 2 minutes, pipette offsupernatant. Add 1 mL of 1×Binding and Washing (B&W; 5 mM Tris-HCl ph7.5, 0.5 mM EDTA, 1 M NaCl) solution and resuspend beads. Place on magnet for 2 minutes to pellet beads and discard supernatant. Resuspend the beads in 500 μL of 1×B&W buffer. Repeat twice for a total of 4 washes. Wash the beads twice in 1 mL of solution A (DEPC-treated 0.1M NaOH, DEPC-treated 0.05 M NaCl) for at least 2 minutes each wash. Then wash the beads in 1 mL of Solution B (DEPC-treated 0.1M NaCl), pellet on magnet and resuspend in 0.5mL Solution B. Pellet beads on magnet and resuspend in 1mL of 2 x Binding and Washing (B&W) solution. Add an equal volume of water with 1 nmol of biotinylated LNA-containing oligo dissolved in it. Incubate for 15 minutes on the Hula shaker at room temperature. Separate the coated beads on a magnet stand for 2-3 minutes, discard the supernatant. Wash the beads 3 times with 1×B&W solution, then resuspend the beads in 1.0 mL of 10mM Tris pH 7.5 or nuclease free water. Store at 4 degrees until ready for use.

[0068] In another example, the amine modified carbon spacer LNA-containing oligonucleotides can be coupled to Dynabeads™ M-270 Carboxylic Acid by resuspending the beads in 25 mM MES (2-Morpholineethanesulfonic acid hydrate) buffer and 700 pmol of 5’ amine-modified oligo with a carbon spacer in 25 mM MES added. The tube is mixed and 300 uL of 10 mg / mL of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) made in cold 100 mM MES is added. After 1 hour, an additional 300uL of 10 mg / mL EDC is added and incubation is continued for another hour. After washing away uncoupled oligos, the beads are incubated for 20 minutes in 50 mM Tris pH 7.5 to react any unreacted carboxylic acid groups and washed and resuspended in PBS with 0.01% tween-20.

[0069] In some embodiments, the nucleic acid(s) isolated via the method disclosed herein can be used for further analysis, e.g., detecting one or more biomarkers in the nucleic acids. The biomarkers can be detected using proper methods known in the art including, without limitation, amplification assay, hybridization assay, and sequencing assay. In certain embodiments, the biomarkers are detected using quantitative RT-PCR, Digital PCR, RNA seq, DNA seq, methylation assay, point mutation assay or CRISPR assay. In certain embodiments, the biomarkers are determined by nucleic acid sequencing.

[0070] The method disclosed herein provides an improvement over the state of the art in several ways. First, we have demonstrated the stronger binding of the LNA containing nucleotides and showed that, when coupled to magnetic beads, enables us to pull down human nucleic acid(s) including mRNA, long noncoding RNA, miRNA or other RNA, DNA or both any form of RNA and DNA simultaneously even in the presence of strong chaotropic salts suchas guanidine thiocyanate (GITC) effectively only when combined with a heating step of between 60 to 90°C for between 1 to 30 minutes. GITC is very commonly used in lysis buffers for its ability to both lysis cells and denature nucleic acid degrading enzymes (Salvi et al., 2005). But chaotropic salts like GITC will also weaken and disrupt hydrogen bonds (Salvi et al., 2005) so that standard oligonucleotides without LNAs lack the ability to efficiently bind to human nucleic acid(s) in the presence of concentrations of 1 or higher molar chaotropic salts like GITC. By using LNA containing oligonucleotides linked to magnetic beads, or other substrates that will allow separation of the nucleic acid(s) from the stool (e.g. the inside of a tube), we can isolate human nucleic acid(s) even in the presence of buffers containing GITC and other chaotropic salts. The ability to isolate nucleic acid(s) directly from preservation / lysis buffers, requires added GITC or GTC with an additional heat-shock step required to eliminate the impact of PCR inhibitors in stool followed by clarification of the supernatant by high-speed centrifugation or filtration to separate stool solids from solubilized molecules prior to hybridization to capture the target molecules. By making these unpredicted modifications we have expanded the scope of conditions and the variety of nucleic acid(s) which can be efficiently captured from crude lysate solutions simultaneously..

[0071] Second, we have shown that LNA bead-based capture has the ability to remove the majority of PCR inhibitors even in very challenging matrices with abundant PCR inhibitors like stool. PCR following nucleic acid(s) purification is a very common downstream requirement and removing PCR inhibitors makes the purified nucleic acid(s) compatible with a wider range of downstream assays / experiments and ensures the detected signal is representative of nucleic acid(s) present in the sample.

[0072] Third, the amount of nucleic acid(s) that is obtained using the method disclosed herein is 2-4 fold higher in quantity and concentration than isolated by other existing approaches. This is a substantial advantage for applications where there is little nucleic acid(s) present such as the case for host or eukaryotic nucleic acid(s) in stool and for when sample material may be limited or expensive including clinical samples.

[0073] Fourth, the ability to directly isolate eukaryotic nucleic acid(s) from complex matrices away from eukaryotic ribosomal RNA, mitochondrial RNA or any bacterial or viral RNA is essential for doing RNA-seq experiments where every RNA molecule will be sequenced. The usual enrichment techniques by rRNA depletion result in the loss of much of the targeted nucleic acid(s), requiring additional time and expense to carry out.

[0074] The method disclosed herein has been demonstrated to provide a substantial improvement for host nucleic acid(s) capture directly from difficult matrices such as stool.

[0075] Kits

[0076] In another aspect, the present disclosure provides kits for use in the methods described above. The kits may comprise any or all of the reagents to perform the methods described herein. In certain embodiments, the kit comprises reagents for making a solution comprising a chaotropic salt; and reagents for making a substrate coupled to a LNA oligonucleotide complementary to the nucleic acid(s)A to be isolated. In some embodiments, the reagents for making a substrate coupled to an oligonucleotide comprises a substrate, a LNA- containing oligonucleotide, and a nucleic acid-conjugation reagent. In some embodiments, the kit comprises primers for detecting a biomarker in a stool sample. In certain embodiments, the kit further comprises an agent for amplifying the target nucleic acid using the primers. The kits may comprise buffers, reagents, binding agents, catalysts, or other chemicals or biological molecules necessary for conducting a chemical or enzymatic reaction. The kits of the present disclosure may further comprise instructions for using the components of the kit or for implementing any of the methods and processes described herein.

[0077] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. EXAMPLES Example 1

[0078] This example illustrates the quantification of GAPDH, a highly expressed human gene, as a proxy for the amount of human mRNA recovered by an exemplary method and kit described herein.

[0079] The protocol we used to test LNA beads in stool is as follows: Wash the beads 1 x with DNA RNA Shield buffer (Zymo Research), then resuspend in 0.5 ml DNA RNA Shield buffer. Leave the bead in the buffer on a i magnet until ready to use then remove the DNA RNA Shield buffer and resuspend the beads to the clarified stool sample. Stool aliquots, frozen at -80C, were resuspended in DNA RNA Shield buffer directly in the cryogenic vial. The DNA RNA Shield was added at a ratio of 1 mL per 250 mg of stool while the stool was still frozen. Samples were vortexed for 1 minute to aid in resuspension of the stool. Then 6 molar guanidine thiocyanate (GTC) or guanidine isothiocyanate (GITC) was added to a final concentration of 1.5 molar and the room-temperature samples were placed in a heater shaker to heat-shock the samples at 75 C for 5 minutes to ensure lysis of human cells and denaturation of inhibitors.Clarification of stool samples is required and was carried out by centrifugation for 4 minutes at 13,000 × g. The clarified supernatant was then transferred into a tube containing prepared magnetic beads covalently bound to LNA probes. The magnetic beads with coupled LNA capture probes were resuspended in the clarified supernatant and loaded into a 96 deep well microtiter plate that was processed on an automation magnetic processing instrument (KingFisher Apex) set up to run a mRNA extraction program similar to that of the ThermoFisher Scientific mRNA Direct Kit with a few modifications. This process may be conducted with or without automation.

[0080] To make sure that we correctly coupled the LNA-capture probe magnetic beads, we compared the ability of our LNA-coupled magnetic beads to commercially available mRNA direct magnetic beads to capture a synthetic sample oligonucleotide that was identical in sequence to the target amplicon nucleic acid (a “gene block”) with a 3’ polyA tail. As shown in FIG.1, both the commercial kit and the poly dT LNA coupled magnetic beads captured the target with similar efficiency. Example 2

[0081] This example illustrates that heating the stool sample in GITC buffer would improve lysis or inhibitor removal using the method and kit described herein. As shown in FIG. 2, following the normal extraction protocol results in worse performance by LNA-mediated capture than a commercially available mRNA capture kit. FIG. 3, a heat-shock treatment of the stool sample in GITC buffer showed an improvement in Ct values. A brief, 5 minute heat shock of the sample to 75°C during the lysis step provided a further ~ 1 Ct improvement or a doubling of mRNA detected by PCR performance for the direct mRNA capture technique when using LNA-containing capture probes, but not when using standard poly dT oligo capture probes. The heating step was instrumental in further removal of inhibitors from the isolated mRNA, allowing 8 times increased input into PCR with a log-linear increase in performance and without degradation of PCR performance as shown in FIG.3.

[0082] We also showed that the improved mRNA direct capture using LNA-containing probes worked across multiple different stool samples (see FIG. 4). We determined that the heat shock step alone was not enough to give the improvement seen in GITC + heating lysis, and that adding GITC, to the already lytic preservation buffer, was required for optimal detection of mRNA determined by measuring low Cts. We tested 6 different stool samples using an automated extraction instrument (KingFisher Apex). Frozen aliquots were resuspended in DNA RNA Shield buffer (Zymo Research), GITC was added and the samples were heated to 75°C for 5 minutes. After heating the samples were shaken for 10 minutes atroom temperature on a Hula Shaker. Samples were then processed on the KingFisher Apex using the buffers and program from ThermoFisher for the mRNA direct capture kit with modifications for capturing the magnetic beads as the biotin magnetic beads pulled to a magnet slower than the beads that came with the kit.

[0083] In addition, we show that by using either target specific probes or polydT probes we can capture the most signal from a set of targets where one is mostly intact and the other is partly fragmented FIG.5. Here GAPDH is mostly intact and the polydT probe captures more signal than a target specific probe, while for the CECAM target the target capture probe can capture more signal than the polydT probe. This is the case when the mRNA is fragmented so the polyA tail is separated from the region where the TaqMan assay is located. Example 3

[0084] This example demonstrates an exemplary method described herein, named ECB (Enrichment Capture By hybridization) Extract, with the following key components: 1) using locked nucleic acids (LNAs)17in the probe design to enhance the binding affinity between the probe and nucleic acid; 2) a customized stool lysis and probe hybridization buffer which has improved capability to dissolve the stool mucus and food debris as well as denature the large amount of nuclease in the stool; 3) customized oligos for the capture of both targeted and poly dT nucleic acids from the crude lysate, which enables high capture efficiency for both fragmented and intact transcripts.

[0085] As demonstrated in the following examples, our ECB Extract method results in a number of improvements over existing stool RNA extraction technology including the capability to process large amounts of stool extract, removing PCR inhibitors, and enriching both fragmented and intact nucleic acid.

[0086] Phenol Extraction Method

[0087] The “gold standard” for nucleic acid stool extraction is phenol chloroform-based extraction. We used the E.Z.N.A. Kit from Omega Biotek (Cat number R6828-02) for phenol chloroform extractions according to the manufacturer's instructions with minimal modifications. Briefly, raw stool samples were lysed. Phenol was added, and the sample was vortexed and incubated at 65°C for 10 minutes. Chloroform was added, and the sample was mixed and centrifuged. The aqueous layer was transferred to a new tube, and Buffer RB and absolute ethanol were added. The sample was then spun through a HiBind column. When scaling up the stool input into extraction, this process was repeated several times to accommodate the full 1-gram sample. Then, DNase 1 was added to the column and the column was incubated for 30 minutes at room temperature. The flow through was discarded. Thecolumn was washed twice and eluted using 55 uL of DEPC-treated water. Finally, the sample was purified on a Zymo-spin ™ III-HRC column by centrifugation.

[0088] Silica Bead Method

[0089] The variant of the “Boom method” (Silica bead method) was done as follows: to an aliquot of frozen supernatant processed as above 1mL of nuclease-free water was added, and the suspension was centrifuged. Proteinase K digestion was done for 20 minutes, then Lysis binding buffer (Viral NA kit Thermo Fisher) and isopropanol were added, and the sample was mixed. SILANE magnetic beads were added and the sample was shaken for 15 minutes. The beads were pelleted on a magnet, and the sample was washed once with MagMax Wash buffer 1. Beads were then washed with 450 μL wash buffer 2 twice and air dried. Elution was into nuclease-free water at 70 °C. Extractions of larger stool input volumes had all volumes scaled up to match the stool volume.

[0090] ECB Extract Method

[0091] Coupled magnetic beads were added to the supernatant from above and incubated to capture the mRNA. Beads were washed multiple times in Wash Buffer A (Thermo Fisher mRNA DIRECT ™) and then washed with Wash Buffer B. RNA was eluted on a Thermomixer for 2 minutes. Extractions done with larger stool volumes had all volumes scaled up to match the larger stool input. The stool lysis / hybridization buffer process is as follows: take 1 gm stool resuspend in 4 mL DNA RNA shield (Zymo Cat. No. R1100-250), add 2mL 6 M GITC and 2 mL Inhibit Ex (Qiagen Cat. No.19593), mix and heat to 70 C for 5 min, then shake at room temperature for 10 minutes to completely dissolve the stool, lyse the cells, and free the RNA. Example 4

[0092] This example illustrates that ECB Exact method can be used to process large amounts of stool extract.

[0093] One difficulty faced by stool extraction methods is the high amounts of PCR inhibitors existing in the purified nucleic acids. Increased stool input into extraction reactions would concentrate the signal and improve the sensitivity of PCR assay if PCR inhibitors are efficiently removed. We tested the ECB Extract method to determine if it remains PCR inhibitor-free when scaling up the stool input into extraction. As shown in FIG 6A-6C, ECB Extract method demonstrated a linear decrease in Ct values with increased stool input into the extraction while the phenol method (Omega E.Z.N.A. Kit) or silica bead extraction (Boom et al., 1990) did not shown a linear decrease, indicating the presence of PCR inhibitors in the eluate from the phenol method and silica bead method.

[0094] Similarly, the ECB Extract performed better than the mRNA DIRECTTMkit (Thermo Fisher) in scaling up samples. The mRNA DIRECTTMkit worked well for some samples but could not remove many of the PCR inhibitors whose presence precludes adding more stool into extraction or more eluate into PCR reactions for improved detection (see Table 2).

[0095] Table 2: Comparison of ECB extract method with mRNA DIRECTTMKit

[0096] The ECB Extract method allows us to sample larger stool volumes, isolate both more target molecules from an extraction and generate an eluate that is more representative of the human RNA in a stool sample into the extraction.

[0097] The ECB Extract method also allows for more stringent hybridization and wash conditions resulting in lower Cts, and improved delta Ct when adding more eluate into the same volume of PCR reaction. Doubling the amount of template into a PCR reaction should give a 1 Ct decrease if all the inhibitors have been removed. If the inhibitors have not been removed, adding more eluate into PCR reactions will not improve the Cts and often will generate higher Cts. Comparing the ECB Extract method to the phenol extraction kit and the silica bead method shows that the ECB Extract method consistently improved the Cts with increased eluate input into PCR as if it removes more of the PCR inhibitors than either of the comparator methods in both normal or clinical stool samples. The ECB Extract method amplification efficiencies were high and consistent across six samples, with slopes ranging from -1.0061 to -1.0963 and R² values 0.9947 (see Table 3).

[0098] Table 3. Increasing amounts of extract eluate from six clinical stool samples were used to generate 5-point curves and calculate the slope and the R2. The slope close to -1 demonstrate a strong tendency for Ct values to improve as more extract eluate is added to the PCR reaction.Example 5

[0099] This example demonstrates that ECB Extract is better at removing PCR inhibitors from stool extractions than commercial stool extraction kits.

[0100] The extraction processing schematic is shown in FIG.7A. Increasing amounts of stool up to 1 g were added to the extractions while scaling up other extraction steps as needed. Final elution volume was kept the same for all extraction methods. PCR targets were GAPDH or TGFB1. FIG. 7B and 7C shows the Cts for 2 mRNA targets extracted by Phenol method, Silica beads or ECB Extract. A 0.5 g stool extraction was run for 6 clinical samples using each extraction method. After extraction increasing amounts of eluate were added to RT-qPCR reactions and the resulting Cts measured by Taqman assay using our mRNA-specific Taqman assay for GAPDH (FIG.7D-7F).

[0101] Another measure of the level of inhibitors in extracted stool samples is how well MS2 phage, spiked into the eluate, can be amplified by PCR. To determine if the improvements in Cts we see with increasing eluate input into PCR and by increased stool into extraction, from different extraction methods, are due to the presence of less inhibitors in the ECB Extract method, we spiked MS2 phage into the eluates of 20 clinical samples extracted by either the Phenol method or ECB Extract. The MS2 PCRs show that many of the eluates from the Phenol method samples still contain moderate to high levels of PCR inhibitors while the eluates from ECB Extract samples have no detectable PCR inhibitors in them (FIG.7G). Example 6

[0102] This example demonstrates the use of customized oligos for the capture of both targeted and poly dT nucleic acids from the crude lysate.

[0103] To be certain that a PCR assay is mRNA-specific requires the primers or probe to cross an intron splice junction or to place the primers on exons that are separated by an intron of at least 1000 base pairs. As a result, mRNA specific assays often cannot be located close to the polyA tail. Human RNA isolated from harsh environments, such as stool, is often fragmented or partially degraded. To consistently capture the signal from the potentially fragmented RNA in stool samples of varying quality we designed target capture probes to selectively isolate the mRNA fragments containing our assay sequences. First we compared the fragmentation level of 5 different normal or CRC clinical samples using a 3’5’ assay which measures how often mRNA molecules of the gene are intact by using a 2 step PCR where poly dT is the primer used to create a cDNA (FIG.8A). Then we compared the ability of the bead types (poly dT, individual target capture beads where each bead has the probe for a single target, or combination beads with poly dT + target capture probes coupled to the same bead (ECB Extract)) to isolate slightly or severely fragmented mRNA from either GAPDH or. We find that the beads with poly dT or ECB Extract beads do very well to capture all the signal when the mRNA is not or only slightly fragmented (FIG. 8B and 8C, samples 28 and 29). For samples with higher levels of fragmentation either the target capture or the ECB Extract beads do best FIG. 8B and 8C, samples 30, 31 and 32. Overall, combining target-specific capture beads + poly dT on a single bead consistently isolates as much as or more mRNA signal as the other bead types from stool samples regardless of their mRNA fragmentation levels (FIG.8B and 8C). Example 7

[0104] This example demonstrates the comparison of diagnostic performance using different extraction methods.

[0105] To determine if increased sensitivity of detection translates to improved diagnostic performance, we tested the ECB Extract method, FIT, and a “gold standard” phenol method on 73 clinical samples (CRC=27, APL=21, hyperplastic polyps + normal colonoscopy = 25) and 12 genes (11 biomarkers and 1 normalization gene) we have previously shown were overexpressed in CRC stool samples 23, 27, 28.

[0106] Direct comparison on the same samples for their CRC diagnostic performance show that the ECB Extract samples had higher AUC for 10 of the 11 individual biomarkers tested, (FIG. 9A), with a number of genes having substantially improved performance using ECB Extract. For example, MMP7 using ECB Extract has an AUC of 0.96 compared to 0.79 AUCusing Phenol method while IL11 has an AUC of 0.91 compared to 0.8 using the Phenol method. Overall, the average increase in AUC comparing ECB Extract to Phenol method was 0.0584. . FIG.9B and 9C: CRC diagnostic ROC curves for each gene are plotted for Phenol method and ECB Extract. It is clear that the ROC curve for the ECB extract method achieves a higher true positive rate at a lower false positive rate than the Omega methos curve does FIG.9D: Plotted for each gene is APL AUC comparing Phenol method (Y-axis) with ECB Extract (X-axis). Genes below the diagonal represent genes with better APLA diagnostic performance using ECB Extract compared to Phenol method. FIG.9E and 9F: APL diagnostic ROC curves for each gene are plotted for Phenol method and ECB Extract.

[0107] The AUC performance of the ECB Extract extraction also showed pronounced improvements for APL detection (9 out of the 11 biomarkers had better diagnostic performance using ECB Extract) (FIG. 9D). Genes such as MMP7 had an APL AUC of 0.77 versus 0.62 using the Phenol method. In general, the average increase in APL AUC comparing ECB Extract to the Phenol method was 0.06.

[0108] All 11 biomarkers were then combined into a panel, and gene panel's diagnostic performance on this set of 27 CRC, 21 APL, and 25 Normal + NAPL samples was tested using random forests and five-fold cross validation. The panel's performance gives a sensitivity of 96% for CRC and a 50% sensitivity for APLAA) at a specificity of 96% (see Table 4).

[0109] Table 4. Diagnostic Performance Comparison of Phenol method with ECB Extract

[0110] We then compared the diagnostic improvement using ECB Extract by comparing how “binary” the gene expression of a biomarker was. Using a previously published patient cohort which used the Phenol method extraction18. Genes were categorized as “binary” if gene expression was detected in less than 10% of control samples but with significant expression in many CRC samples (CRC AUC > 0.7) (MMP3, NKD2 and IL11). Moderately binary genes were genes with detectable expression in a minority of control samples, i.e., 10-50% of control samples but with significant expression in many CRC samples (CRC AUC > 0.7) (MMP7,PPBP and TCN1). Non-binary genes were genes with detectable expression in most, i.e., > 50% of control samples but with significantly higher expression in many CRC samples (CRC AUC > 0.7) (OLR1, CXCL8, TGFBI, MYC, HBA). There was a strong tendency for binary genes and moderately binary genes to have more significant improvements in diagnostic performance using ECB Extract than the non-binary genes. The binary genes had an average improvement in CRC AUC using ECB Extract of 0.1 with an average APL improvement of 0.08. Moderately binary genes had an average CRC AUC improvement of 0.06 with an average APL AUC improvement of 0.09. Non-binary genes had an average CRC AUC improvement of 0.04 with an average APL AUC improvement of 0.02 (See Table 5).

[0111] Table 5. AUC of Biomarkers for CRC and AA.Example 8

[0112] This example demonstrates the clinical study using ECB Extract method.

[0113] Given the improved diagnostic performance observed using ECB Extract, we increased the number of biomarkers and clinical samples tested using ECB Extract. An additional 5 genes were tested on 173 additional clinical samples. The additional genes added were some of the top performing genes from previous studies we have performed18,19. A total of 16 gene’s expression was quantified using ECB Extract on 246 clinical stool samples (See below for list of genes and each gene's performance).

[0114] Table 6. LNA capture probes (symbol “+” represents that the previous nucleotide is a LNA)

[0115] Table 7. List of 16 genes and diagnostic performance for CRC and APL detection.

[0116] To obtain a small enough gene subset to form a putative clinical panel, biomarker selection was performed to get a subset of genes with at least as good performance as all 16 genes combined. PromptBio, an AI-driven commercial software tool20was used to select the most informative subset of genes. A 8 gene panel was obtained (MMP7, TGFBI, NKD2, EPHX4, IL11, PPBP, IGF2 and HBA). HBA represents the combined gene expression of HBA1 and HBA2 hemoglobin subunit genes. In a previous manuscript21and patent we showed HBA can serve as a replacement for FIT in detecting occult blood in stool. The gene panel's diagnostic performance was tested using random forests and five-fold cross validation. The combined panel's performance gives a sensitivity of 95.9 % for CRC (N=63) and a 54.5 % sensitivity for APLAA (N=42) at a specificity of 95.1 % (for combined NEG + NALP; NAPL (N=45), NEG=96 (Negative Coloscopy (N=58) + HP (N=38)) (Table 8). As a point of comparison FIT performance was 78% for CRC sensitivity, 13.8% sensitivity for APL detection at a specificity of 95.6% (control group consists of NAPL+NEG colonoscopy findings). The demographic and clinical subcategory performance of the 8 gene panel are given in Tables 9- 11.

[0117] Table 8. Summary of the CRC Test Performance

[0118] Table 9. Baseline Demographics for the Average-risk Cohort

[0119] Table 10. The xCRC test CRC Sensitivity by Colonoscopy Categories, Compared to Independent FIT CRC Sensitivity

[0120] Table 11. The xCRC test APL Sensitivity by Colonoscopy Categories Compared to Independent FIT APL Sensitivity

[0121] The signal amplification achieved by RNA could potentially provide an advantage in detection of CRC and APL over using DNA based biomarkers if the tools to extract and purify host RNA are robust enough to consistently isolate easily detectable quantities of inhibitor-free RNA. Improved RNA extraction also allows for the use of RNA biomarkers with improved differential expression and enhanced specificity for CRC and APL which are not reliably detected by current extraction methods. We have devised an RNA isolation method, ECB Extract, that demonstrates improved isolation of human RNA from stool. The improvements include: fewer inhibitors in the eluate, enrichment of target RNA for both fragmented and intact RNA molecules, semi-automatability, scalability to larger stool input volumes, and ultimately, improved clinical performance of biomarkers. The improved lysis,capture of RNA, and inhibitor removal we can achieve with ECB Extract allows us to concentrate larger amounts of stool in the extraction process and larger amounts of eluate into our PCR assays. This improves the sensitivity of detection for our biomarkers resulting in improved diagnostic performance compared to the gold standard phenol-chloroform extraction method.

[0122] In addition to larger amounts of stool, ECB Extract also pairs polyA selection with target capture probes multiplexed on the same bead. Our data shows that polyA capture performs very well at capturing intact transcripts, performing better than target capture does. This may be due to the target sequence, when present in intact molecules, being partially blocked due to RNA secondary structure. In addition, the target sequence is commonly towards the center of the transcript and hence capturing the sequence requires bending a complete transcript and pulling it close to the bead surface where it may have steric interactions with other large transcripts in close proximity. For intact transcripts, the polyA tail will likely not be heavily involved in RNA secondary structure and will therefore be accessible to be captured. In addition, since the polyA tail is attached to the end of the transcript, pulldown to the bead surface does not require overcoming a high degree of steric hindrance. When the transcript is fragmented, target capture outperformed polyA selection.

[0123] As we have shown, the degree of host RNA fragmentation is highly variable from sample to sample. While polyA capture may work very well for some samples for others it will not and vice versa for target capture approaches. By combining both polyA and target capture probes on the same bead, ECB Extract can obtain the best of both worlds.

[0124] Another benefit of our approach is the ability to capture target RNA sequences in a concentrated chaotropic environment. Chaotropic salts such as guanidinium isothiocyanate (GITC) are commonly used to lyse cells and inactivate nucleases. However, chaotropic salts also weaken hydrogen bonds so traditional hybridization capture approaches first require bulk purification of nucleic acids before hybridization capture takes place. Because we use LNAs in our capture probes we can perform hybrid capture directly from the crude lysate which contains a high concentration of GITC. This is due to the much stronger hydrogen bonding that LNA nucleotides exhibit. In ECB Extract we add high concentrations of GITC to aid in lysing cells in the difficult stool environment and to inactivate nucleases. Because we use capture probes containing LNAs, we are able to directly capture from the crude lysate without requiring bulk purification of nucleic acids which increases our RNA yield and simplifies the extraction procedure.

[0125] While existing extraction techniques are suitable for highly expressed stool mRNA biomarkers, using only highly expressed genes limits the pool of informative, clinically useful, RNA biomarkers. For many of the biomarkers tested, in particular the “binary” biomarkers, using a more sensitive extraction technique, such as ECB Extract, substantially improved sensitivity of lesion detection with little if any loss of specificity. This improvement in sensitivity boosted the number of clinically useful biomarkers. In particular, for APL detection, using a traditional phenol-chloroform approach 1 out of the 11 biomarkers tested had an AUC > 0.7 (TGFBI). However, when using ECB Extract, 5 of the biomarkers (MYC, MMP7, TGFBI, HBA, PPBP) had an APL AUC > 0.7.

[0126] The pattern of binary and moderately binary genes benefiting the most from improved mRNA extraction is not unexpected. These are genes that are undetectable in most control samples but detectable in a significant fraction of colorectal cancer samples. This pattern of expression suggests there are samples for which gene expression is low enough that existing extraction methods do not have the sensitivity to detect them. Increasing the ability to detect the expression of these genes substantially increases the fraction of detectable expression in CRC / APLAA samples while mildly increasing the detection in control samples so overall diagnostic performance is significantly improved.

[0127] For the non-binary genes, expression is already detectable in most control samples, hence increasing the sensitivity of detection will increase the number of detected transcripts for both disease and control samples to roughly the same degree. Overall, the separation between the two groups does not change hence diagnostic performance does not measurably improve with increased sensitivity of detection for the non-binary genes.

[0128] In general, sensitivity for CRC detection was excellent using the 8 gene panel tested on 246 samples. CRC lesion detection was 95.9% at a specificity of 95% (control group consists of NAPL+NEG colonoscopy findings). The location and size of the lesion did not have a strong impact in terms of detection rate but detection of stage I lesions was lower than later stages.

[0129] In contrast to CRC lesion detection, APL detection was more sensitive for lesions located in the rectum compared to more distantly located lesions. In agreement with other published studies 5 lesions with high grade dysplasia were the easiest APL category to detect . Overall, the sensitivity of detection for APL lesions of all categories was 54.5% at a specificity of 95% (control group consists of NAPL+NEG colonoscopy findings).

[0130] The improved isolation method described herein could be applied to allow for non- invasive diagnostics / monitoring for GI related disorders in general including colorectal cancerscreening, inflammatory bowel disease (IBD), Gastrointestinal (GI) infections and other GI- related diseases. Developing enhanced, RNA-derived stool-based, tests for population screening with improved sensitivity and specificity for both CRC and APL would be an important advance for early detection of this consequential disease.References 1. Ballantyne, K.N., van Oorschot, R. a. H., Mitchell, R.J., 2008. Locked nucleic acids in PCR primers increase sensitivity and performance. Genomics 91, 301–305. 2. Blandino, G., Dinami, R., Marcia, M., Anastasiadou, E., Ryan, B.M., Palcau, A.C., Fattore, L., Regazzo, G., Sestito, R., Loria, R., Díaz Méndez, A.B., Cappelletto, M.C., Pulito, C., Monteonofrio, L., Calin, G.A., Sozzi, G., Cheong, J.K., Aharonov, R., Ciliberto, G., 2023. The new world of RNA diagnostics and therapeutics. J. Exp. Clin. Cancer Res.42, 189. 3. Byron, S.A., Van Keuren-Jensen, K.R., Engelthaler, D.M., Carpten, J.D., Craig, D.W., 2016. Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Nat. Rev. Genet.17, 257–271. 4. Dan, S., Ungar, B., Ben-Moshe, S., Bahar Halpern, K., Yavzori, M., Fudim, E., Picard, O., Abitbol, C.M., Harnik, S., Barshack, I., Kopylov, U., Ben-Horin, S., Itzkovitz, S., 2023. Distal Fecal Wash Host Transcriptomics Identifies Inflammation Throughout the Colon and Terminal Ileum. Cell. Mol. Gastroenterol. Hepatol.16, 1–15. 5. DynabeadsTM mRNA DIRECTTM Purification Kit [WWW Document], n.d. URL https: / / www.thermofisher.com / order / catalog / product / 61012 (accessed 4.24.24). 6. He, K., Fujiwara, H., Zajac, C. et al. A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR. Sci Rep 9, 5599 (2019). 7. Hou, K., Wu, ZX., Chen, XY. et al. Microbiota in health and diseases. Sig Transduct Target Ther 7, 135 (2022).. 8. Jacobsen N, Nielsen PS, Jeffares DC, Eriksen J, Ohlsson H, Arctander P, Kauppinen S. Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture. Nucleic Acids Res. 2004 Apr 19;32(7):e64. PMID: 15096560; PMCID: PMC407836. 9. Kasırga E. The importance of stool tests in diagnosis and follow-up of gastrointestinal disorders in children. Turk Pediatri Ars. 2019 Sep 25;54(3):141-148. doi: 10.14744 / TurkPediatriArs.2018.00483. PMID: 31619925; PMCID: PMC6776453.10. Ryan L, Wong Y, Dwyer KM, Clarke D, Kyprian L, Craig JM. Coprocytobiology: A Technical Review of Cytological Colorectal Cancer Screening in Fecal Samples. SLAS TECHNOLOGY: Translating Life Sciences Innovation.2021;26(6):591-604. 11. Reck, M., Tomasch, J., Deng, Z., Jarek, M., Husemann, P., Wagner-Döbler, I., 2015. Stool metatranscriptomics: A technical guideline for mRNA stabilization and isolation. BMC Genomics 16, 494. 12. Ungar, B., Yavzori, M., Fudim, E., Picard, O., Kopylov, U., Eliakim, R., Shouval, D., Levin, Y., Savidor, A., Ben-Moshe, S., Manco, R., Dan, S., Egozi, A., Bahar Halpern, K., Mayer, C., Barshack, I., Ben-Horin, S., Itzkovitz, S., 2022. Host transcriptome signatures in human faecal-washes predict histological remission in patients with IBD. Gut 71, gutjnl-2021- 325516. 13. Zhao, S., Zhang, Y., Gamini, R., Zhang, B., von Schack, D., 2018. Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion. Sci. Rep.8, 4781. 14. Barnell, E. K. et al. Multitarget Stool RNA Test for Colorectal Cancer Screening. JAMA 330, 1760–1768 (2023). 15. Ryan, L. et al. Coprocytobiology: A Technical Review of Cytological Colorectal Cancer Screening in Fecal Samples. SLAS Technol.26, 591–604 (2021). 16. Koga, Y. et al. Improved Recovery of Exfoliated Colonocytes from Feces Using Newly Developed Immunomagnetic Beads. Gastroenterol. Res. Pract.2008, 605273 (2008). 17. Singh, S. K., Koshkin, A. A., Wengel, J. & Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun.455–456 (1998) doi:10.1039 / A708608C. 18. Hansen, L. et al. Identification of Novel mRNA Biomarkers with Improved Performance for Colorectal Cancer Screening from a Large Multicenter Screen. (In Preparation) 19. Liu, H. et al. Bioinformatic screen with clinical validation for the identification of novel stool based mRNA biomarkers for the detection of colorectal lesions including advancedadenoma. Sci. Rep.15, 29397 (2025). Zhang, M. et al. PromptBio: A Multi-Agent AI Platform for Bioinformatics Data Analysis.2025.07.05.663295. Wenying, P. et al. Fecal HBA mRNA as an Alternative to the Protein-based FIT Assay for Colorectal Cancer Screening: A Computational and Clinical Validation Study. (In Preparation)

Claims

WHAT IS CLAIMED IS:

1. A method of isolating eukaryotic nucleic acids from a stool sample comprising: a) obtaining a stool sample from a subject, wherein the stool sample comprises at least a nucleic acid from the subject; b) incubating the stool sample in a solution containing a chaotropic salt; c) heating the stool sample to a temperature between 60°C and 90°C; d) incubating the stool sample with a substrate coupled to a locked nucleic acid (LNA) oligonucleotide, wherein the LNA oligonucleotide comprises a sequence complementary to the nucleic acid, thereby allowing the RNA to hybridize to the LNA oligonucleotide; and e) isolating the nucleic acid hybridized to the LNA oligonucleotide.

2. The method of claim 1, wherein the solution does not contain phenol.

3. The method of claim 1, wherein the stool sample is incubated with the chaotropic salt at a concentration of about 1.5M in step (b); optionally, wherein the stool sample is heated for 1 to 15 min minutes in step c).

4. The method of claim 1, wherein the solution containing the chaotropic salt is a preservation buffer or lysis buffer; optionally, the solution contains lithium chloride and sodium dodecyl sulfate; optionally, the chaotropic salt is guanidinium isothiocyanate (GITC) or guanidinium thiocyanate (GTC).

5. The method of claim 1, wherein the LNA oligonucleotide comprises poly- deoxythymidine (polydT); optionally, the LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs: 1-9.

6. The method of claim 5, wherein the substrate is couple to a second LNA oligonucleotide comprising a sequence complementary to an internal sequence of the nucleic acid; optionally, the second LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs:

7. The method of claim 1, wherein the substrate is a magnetic, a paramagnetic, or a super paramagnetic bead.

8. The method of claim 1, wherein the oligonucleotide is coupled to the substrate via a biotin modified 5’ end, a carboxyl amine coupling or any one of the usual ways of coupling nucleic acids to surfaces.

9. The method of claim 1, wherein the oligonucleotide is coupled to the substrate via a 5’ amine modified end with a carbon spacer.

10. The method of claim 1, wherein the method is executed via an automation.

11. A kit for isolating a eukaryotic nucleic acid from a stool sample comprising: reagents for making a solution comprising a chaotropic salt; and reagents for making a substrate coupled to a LNA oligonucleotide comprising a sequence complementary to the eukaryotic nucleic acid.

12. The kit of claim 11, wherein the LNA oligonucleotide comprises poly-deoxythymidine (polydT); optionally, the LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs: 1-9.

13. The kit of claim 12, further comprising reagent for making the substrate coupled to a second LNA oligonucleotide comprising a sequence complementary to an internal sequence of the eukaryotic nucleic acid; optionally, the second LNA oligonucleotide has a sequence of at least 90% identity to any of SEQ ID NOs: 10-25.

14. The kit of claim 11, wherein the substrate is a magnetic, paramagnetic, or super paramagnetic bead.

15. The kit of claim 11, further comprising a preservation buffer for collecting the stool sample and / or a lysis buffer; optionally, the solution contains lithium chloride and sodium dodecyl sulfate; optionally, the chaotropic salt is guanidinium isothiocyanate (GITC) or guanidinium thiocyanate (GTC).

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