Method and kit for isolating and sequencing nucleic acid from skin samples

Abstract

The present disclosure relates to methods for and kits for isolating and sequencing nucleic acid from skin samples.

Description

[0001] METHOD AND KIT FOR ISOLATING AND SEQUENCING NUCLEIC ACID FROM SKIN SAMPLES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 681 ,385, filed, filed August 9, 2024, the disclosure of which is incorporated by reference in its entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] The present disclosure relates to methods for and kits for isolating and sequencing nucleic acid from skin samples.

[0006] BACKGROUND

[0007] The skin serves as a first line of defense against many pathogens and is marked by a consistent immune cell presence that makes inflammatory skin conditions extremely prevalent in dermatology. These conditions range in severity from uncomfortable itching and rashes to chronic skin conditions including atopic dermatitis (AD), psoriasis, and other related conditions (e.g., eczematous dermatitis). For example, while AD and psoriasis are distinct diseases, both are characterized by inflamed, scaling, and thickened skin lesions that vary in intensity and body area surface affected. AD is a relapsing skin disease that is estimated to affect approximately 20% of children and 7-14% of adults. While most patients are diagnosed before the age of 5, AD can persist throughout adulthood in up to 50% of cases, and late-onset (age 40+) and very late-onset (age 60+) AD are becoming increasingly common. Overall, psoriasis impacts an estimated 3% of the world’s population. Beyond clinical outcomes, psoriasis and AD have been associated with additional burdens that span beyond physical maladies and into psychological and social aspects of life. These burdens can lead to mental health conditions such as anxiety, depression, and suicidal ideation. Importantly, patients with related conditions, such as eczematous dermatitis, that is not diagnosed as atopic dermatitis are also significantly impacted by the burden of their skin disease.

[0008] There is a need for improved methods and kits to better isolate nucleic acid from non- invasively collected skin samples in order to help better diagnose and treat patients having a skin disease or disorder. The methods and kits disclosed herein help better diagnose and treat patients having a skin disease or disorder, which could result in reduced cost burden, improved patient responses, and reduced risks associated with continued treatment.

[0009] SUMMARY

[0010] The methods described herein provide a technique used to collect, transport, extract, and sequence nucleic acid (e.g. RNA or DNA) from skin samples. In an embodiment, the methods extract nucleic acid on the KingFisher® system and sequencing is performed on the Ion GeneStudio™ S5 Prime next-generation sequencing (NGS) system.

[0011] The methods as disclosed herein were adapted from the manufacturer’s protocols for both RNA extraction and next-generation sequencing procedures. However, significant modifications were surprisingly discovered that were required to adapt and optimize to the skin sample and experimental conditions.

[0012] In one aspect, this disclosure provides methods comprising:

[0013] (a) obtaining a skin sample;

[0014] (b) extracting nucleic acid from the sample, wherein extracting comprises: i) lysing the skin sample using a homogenizer to generate a homogenized sample; ii) mixing the homogenized sample for about 5 minutes at about 1 150 rpm; iii) adding isopropanol to each mixed homogenized sample and mixing for about an additional 2 minutes at about 950 rpm; iv) combining the mixed homogenized sample comprising isopropanol with beads comprising a nucleic acid binding surface and incubating for about 5 minutes at about 950 rpm to bind the nucleic acid to the beads; v) washing the beads and treating the washed beads with a DNase; vi) washing the DNase treated beads; and vii) eluting the nucleic acid from the beads to generate an isolated nucleic acid sample.

[0015] In certain embodiments of the methods disclosed herein, the skin sample is stored in an extraction buffer for up to about 96 hours at room temperature.

[0016] In certain embodiments of the methods disclosed herein, the method further comprises quantitating the isolated nucleic acid sample.

[0017] In certain embodiments of the methods disclosed herein, the nucleic acid comprises DNA, RNA, or both DNA and RNA. In an embodiment, the nucleic acid comprises RNA.

[0018] In certain embodiments of the methods disclosed herein, the method further comprises reverse transcription of RNA to cDNA. In certain embodiments, reverse transcription comprises incubating at about 25°C for about 10 minutes, then at about 42°C for about 60 minutes, then at about 85°C for about 5 minutes, and finally at about 4°C for up to about 18 hours, or optionally at -20°C for up to one year.

[0019] In certain embodiments of the methods disclosed herein, the isolated nucleic acid sample is treated to remove contaminants.

[0020] In certain embodiments of the methods disclosed herein, the methods further comprise amplifying a population of targets from the isolated nucleic acid sample to produce a population of amplified targets. In certain embodiments, the methods further comprise ligating barcodes to the population of amplified targets, and sequencing the population of amplified targets.

[0021] In certain embodiments of the methods disclosed herein, the skin sample is obtained by a non-invasive method. In an embodiment, the skin sample is obtained by a curette.

[0022] In certain embodiments of the methods disclosed herein, the skin sample is a FFPE sample. In certain embodiments of the methods disclosed herein, the extraction buffer contains betamercaptoethanol and / or Triton.

[0023] In certain embodiments of the methods disclosed herein, the extraction buffer does not contain thiocyanic acid.

[0024] In certain embodiments of the methods disclosed herein, the sample is stored in the extraction buffer at a temperature that is at least about -80°C for up to about 45 days. In another aspect, this disclosure provides for a kit comprising reagents and instructions for performing the methods as disclosed herein.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows a schematic of a loaded Ion Chef™ Instrument.

[0027] FIG. 2 shows a schematic of a loaded Ion Chef™ reagents cartridge.

[0028] DETAILED DESCRIPTION

[0029] This disclosure provides methods for improved methods and kits to isolate nucleic acid from non-invasively collected skin samples.

[0030] In one aspect, this disclosure provides methods comprising:

[0031] (a) obtaining a skin sample;

[0032] (b) extracting nucleic acid from the sample, wherein extracting comprises: i) lysing the skin sample using a homogenizer to generate a homogenized sample; ii) mixing the homogenized sample for about 5 minutes at about 1150 rpm; iii) adding isopropanol to each mixed homogenized sample and mixing for about an additional 2 minutes at about 950 rpm; iv) combining the mixed homogenized sample comprising isopropanol with beads comprising a nucleic acid binding surface and incubating for about 5 minutes at about 950 rpm to bind the nucleic acid to the beads; v) washing the beads and treating the washed beads with a DNase; vi) washing the DNase treated beads; and vii) eluting the nucleic acid from the beads to generate an isolated nucleic acid sample.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which the claimed invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the methods and kits disclosed or claimed herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the claimed invention will be apparent from the following detailed description.

[0034] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a nucleic acid" means one or more nucleic acids.

[0035] As used herein, ranges and amounts can be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%" and also "5%." The term "about" can also refer to ± 10% of a given value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example. Additionally, "about" or "comprising essentially of can mean a range of up to ±10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of" should be assumed to be within an acceptable error range for that particular value or composition. Unless otherwise clear from context, all numerical values provided herein are modified by the term "about."

[0036] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment disclosed or claimed herein.

[0037] In certain embodiments of the methods disclosed herein, the nucleic acid comprises DNA, RNA, or both DNA and RNA. In an embodiment, the nucleic acid comprises RNA.

[0038] As used herein, the terms "nucleic acid" and "nucleotide" can be used interchangeably to refer to single or double-stranded nucleic acid comprising DNA, cDNA, RNA, mRNA, derivatives thereof, and / or combinations thereof. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. The term "DNA" includes, but is not limited to, genomic DNA and cDNA. The term "RNA" refers to ribonucleic acid that is present in cells that has structural similarities to DNA. RNA is typically single-stranded, and comprises a backbone made of alternating phosphate groups and the sugar ribose, rather than the deoxyribose found in DNA. Different types of RNA that exist in cells can include messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). In certain embodiments, the nucleic acid is DNA. In certain embodiments, the nucleic acid is RNA.

[0039] In certain embodiments of the methods disclosed herein, the method further comprises quantitating the isolated nucleic acid sample.

[0040] As used herein, the term "skin sample" refers to a sample comprising nucleic acid, cells, and / or tissue from the skin of a subject. A skin sample can be normal skin ( / .e. non- lesional or no sign of disease). For example, normal skin can be characterized by a balanced oil-to-water ratio, having a smooth texture, minimal visible pores, an even complexion (neither excessively oily nor dry), and generally doesn't experience frequent breakouts or sensitivity. In certain embodiments, a skin sample can be a skin lesion or suspicious skin lesion ( / .e. any tissue on or in the skin that has abnormal characteristics). For example, a skin lesion or suspicious skin lesion can refer to part of the skin that is inflamed, itchy, scaly or having scaly patches of discolored skin, wheals, red scales, white scales, silvery-white scales, nodules, macules, papules, vesicle, bulla, pustule, bumps, ulcers, sores, crust, excoriation, lichenification, scars, moles, warts, acne, skin tags, cherry angiomas, freckles, solar lentigines, age spots, seborrheic keratosis, and plaques with a greasy, yellow scale. In some embodiments, a suspicious skin lesion can be suspected of being cancer, for example, melanoma, basal cell carcinoma, cutaneous melanoma, or squamous cell carcinoma. In some embodiments, a skin sample comprises the superficial epidermis of lesional and non-lesional skin from subjects with, or suspected of having, atopic dermatitis (eczema), dermatitis, contact dermatitis, seborrheic dermatitis, psoriasis, and mycosis fungoides. The methods disclosed herein are particularly useful for extracting nucleic acid from skin samples comprising a low abundance of nucleic acid, for example samples obtained through a non-invasive sampling method, such as a curette or adhesive film.

[0041] As disclosed herein, a skin sample can be obtained through a non-invasive sampling method. The non-invasive skin sample is obtained from a subject using a method that does not require inserting an instrument through the skin or into a body opening of the subject. For example, the skin sample can be collected by gently scraping the skin with a curette and immediately preserving the sample in a buffer. Curettes come in many handle styles with either round or oval heads of varying sizes from about 1 mm to about 10 mm and can be made of metal (e.g. stainless steel) or plastic. In certain embodiments of the methods disclosed herein, the skin sample is obtained by a non-invasive method. In an embodiment, the skin sample is obtained by a curette. In certain embodiments of the methods disclosed herein, the skin sample is a FFPE sample.

[0042] In certain embodiments of the methods disclosed herein, the skin sample is stored in an extraction buffer for up to about 96 hours at room temperature. Collection of a skin sample can be performed using a sterile, disposable #4mm dermal curette and added to a tube containing an extraction buffer. A typical nucleic acid extraction buffer contains several components that work together to release and stabilize nucleic acid (such as DNA and / or RNA) from cells or tissues. These components can include, but are not limited to, a buffering agent to maintain pH, salts to disrupt cellular structures, detergents to solubilize membranes, and chelating agents and nuclease inhibitors to prevent nucleic acid degradation. In certain embodiments, the extraction buffer comprises beta-mercaptoethanol and / or a detergent (for example Triton X-100). In some embodiments, the extraction buffer does not contain a thiocyanic acid or a thiocyanate. In certain embodiments of the methods disclosed herein, the sample is stored in the extraction buffer at a temperature that is at least about -80°C for up to about 45 days.

[0043] Cell lysis is the process of breaking down a cell's membrane to release its contents (i.e., DNA or RNA). Cell lysis methods can depend on the cell type and the target nucleic acid; however, there are three primary mechanisms for cell lysis: physical, chemical, and enzymatic. Physical methods use mechanical force to disrupt the cell structure. These methods are highly effective for tough cell walls, such as those found in bacteria, yeast, and plants. Physical methods can comprise mechanical homogenization (via, for example, blenders, bead mills, or Dounce homogenizers), sonication, free-thaw cycles, and / or a French Press. Chemical methods can utilize detergents or other chemical agents to solubilize the cell membrane. These are often used for mammalian cells, which have relatively fragile membranes. Chemical methods can comprise detergents (e.g. SDS or Triton X-100), chaotropic salts (e.g. guanidinium thiocyanate or guanidinium hydrochloride), or hypotonic lysis. Enzymatic methods use specific enzymes to break down components of the cell wall or membrane. This is a gentle approach often used for a wide range of cell types. Enzymatic methods can comprise lysozyme, Proteinase K, cellulose, or zymolase.

[0044] In some embodiments of the methods disclosed herein, nucleic acid is extracted using beads that comprise a nucleic acid bind surface. In certain embodiments, the beads are superparamagnetic or paramagnetic beads coated with a specific chemistry that allows them to bind to nucleic acid (DNA and / or RNA). Once the nucleic has bound to the beads, a strong magnet is applied which then allows the removal of cellular debris, proteins, and other contaminants.

[0045] In some embodiments of the methods disclosed herein, the sample is treated with a deoxyribonuclease (a DNase) to degrade DNA in the sample. In some embodiments of the methods disclosed herein, the sample is treated with an RNase to degrade RNA in the sample.

[0046] In certain embodiments of the methods disclosed herein, the method further comprises reverse transcription of RNA to cDNA. In certain embodiments, reverse transcription comprises incubating at about 25°C for about 10 minutes, then at about 42°C for about 60 minutes, then at about 85°C for about 5 minutes, and finally at about 4°C for up to about 18 hours, or optionally at -20°C for up to one year. In certain embodiments of the methods disclosed herein, the isolated nucleic acid sample is treated to remove contaminants. In certain embodiments, the methods disclosed herein comprise converting isolated RNA to cDNA for further analysis. The conversion of RNA to cDNA may be performed using any suitable method known in the art, for example the extracted RNA is converted to cDNA via reverse transcription. A reverse transcriptase enzyme can be used to convert RNA to cDNA. RNA can be reverse transcribed into cDNA using RNA-dependent DNA polymerases such as, for example, reverse transcriptases from viruses, retrotransposons, bacteria, etc. These can have RNase H activity. Suitable reverse transcriptases can include, but are not limited to, AMV reverse transcriptase, MMLV reverse transcriptase, engineered MMLV reverse transcriptase. In an embodiment the reverse transcriptase is a Superscript™ enzyme.

[0047] In certain embodiments of the methods disclosed herein, the methods further comprise amplifying a population of targets from the isolated nucleic acid sample to produce a population of amplified targets. In certain embodiments, the methods further comprise ligating barcodes to the population of amplified targets, and sequencing the population of amplified targets. In certain embodiments, the methods disclosed herein further comprise sequencing of the isolated nucleic acid. As used herein, sequencing can comprise whole genome sequencing, whole exome sequencing, targeted sequencing, and / or metagenomic sequencing. In certain embodiments, Next-Generation Sequencing (NGS) techniques may be used to sequence the isolated nucleic acid extracted from the skin sample. Sample preparation techniques applicable for various NGS approaches are known and have been extensively described, for example in manufacturer instructions for sample preparation kits available for proprietary sequencing technologies of Ion GeneStudio, Illumina, Pacific Biosystems, and Applied Biosystems.

[0048] In another aspect, this disclosure provides for a kit comprising reagents and instructions for performing the methods as disclosed herein.

[0049] Kits can include any combination of components that facilitates the performance of the methods as described herein. A kit that facilitates assessing the expression of the gene or genes may include suitable nucleic acid-based and / or immunological reagents as well as suitable buffers, control reagents, and printed protocols. The article of manufacture may be promoted, distributed, sold, or offered for sale as a unit for performing the methods disclosed herein. Kits that facilitate nucleic acid based methods may further include one or more of the following: specific nucleic acids such as oligonucleotides, labeling reagents, enzymes including PCR amplification reagents such as Taq or Pfu, reverse transcriptase, or other, and / or reagents that facilitate hybridization. In addition, the kits disclosed herein may preferably contain instructions which describe a suitable detection assay.

[0050] Embodiments:

[0051] Embodiment 1 : A method comprising:

[0052] (a) obtaining a skin sample;

[0053] (b) extracting nucleic acid from the sample, wherein extracting comprises: i) lysing the skin sample using a homogenizer to generate a homogenized sample; ii) mixing the homogenized sample for about 5 minutes at about 1150 rpm; iii) adding isopropanol to each mixed homogenized sample and mixing for about an additional 2 minutes at about 950 rpm; iv) combining the mixed homogenized sample comprising isopropanol with beads comprising a nucleic acid binding surface and incubating for about 5 minutes at about 950 rpm to bind the nucleic acid to the beads; v) washing the beads and treating the washed beads with a DNase; vi) washing the DNase treated beads; and vii) eluting the nucleic acid from the beads to generate an isolated nucleic acid sample.

[0054] Embodiment 2. The method of embodiment 1 , wherein the skin sample is stored in an extraction buffer for up to about 96 hours at room temperature.

[0055] Embodiment 3. The method of either embodiment 1 or embodiment 2, wherein the method further comprises quantitating the isolated nucleic acid sample.

[0056] Embodiment 4. The method of any one of embodiments 1-3, wherein the nucleic acid comprises DNA, RNA, or both DNA and RNA.

[0057] Embodiment 5. The method of any one of embodiments 1-4, wherein the nucleic acid comprises RNA.

[0058] Embodiment 6. The method of embodiment 5, wherein the method further comprises reverse transcription of RNA to cDNA.

[0059] Embodiment 7. The method of any one of embodiment 1-6, wherein the isolated nucleic acid sample is treated to remove contaminants.

[0060] Embodiment 8. The method of embodiment 5, wherein reverse transcription comprises incubating at about 25°C for about 10 minutes, then at about 42°C for about 60 minutes, then at about 85°C for about 5 minutes, and finally at about 4°C for up to about 18 hours, or optionally at -20°C for up to one year.

[0061] Embodiment 9. The method of any one of embodiments 1-8 further comprising amplifying a population of targets from the isolated nucleic acid sample to produce a population of amplified targets.

[0062] Embodiment 10. The method of embodiment 9 further comprising ligating barcodes to the population of amplified targets, and sequencing the population of amplified targets.

[0063] Embodiment 11 . The method of any one of embodiments 1-10, wherein the skin sample is obtained by a non-invasive method.

[0064] Embodiment 12. The method of embodiment 11 , wherein the skin sample is obtained by a curette.

[0065] Embodiment 13. The method of any one of embodiments 1-10, wherein the skin sample is a FFPE sample.

[0066] Embodiment 14. The method of any one of embodiments 2-13, wherein the extraction buffer contains beta-mercaptoethanol and / or Triton.

[0067] Embodiment 15. The method of any one of embodiments 2-14, wherein the extraction buffer does not contain thiocyanic acid.

[0068] Embodiment 16 The method of any one of embodiments 2-15, wherein the sample is stored in the extraction buffer at a temperature that is at least about -80°C for up to about 45 days.

[0069] Embodiment 17. A kit comprising reagents and instructions for performing the method of any one of embodiments 1-16.

[0070] EXAMPLES

[0071] The Examples that follow are illustrative of specific embodiments of the claimed invention, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the claimed invention in any way.

[0072] EXEMPLARY PROTOCOL

[0073] 1. Specimen Collection a. Specimen collection is performed using a sterile, disposable #4mm dermal curette and added to a tube containing an extraction buffer (in certain embodiments, the extraction buffer comprises beta-mercaptoethanol and / or a detergent (for example Triton X-100); in certain embodiments the extract buffer does not contain thiocyanic acid). b. Specimens are shipped to a laboratory in the tube containing the extraction buffer at room temp 15-30°C (RT) within 3 days of collection and in an insulated shipping container containing RT gel packs(s).

[0074] 2. Specimen Acceptance Criteria a. The container must be present and intact. b. Two patient identifiers that match the corresponding requisition form must be on the tube. c. The specimen collection date must be no later than 3 days. d. The extraction buffer must not be expired upon arrival.

[0075] 3. Specimen Rejection Criteria a. Specimen Leaking b. Samples that come in frozen c. Specimen not received within defined timeframe d. Broken containers

[0076] 4. Specimen Processing and Storage a. Specimens are stored at -80°C between 6 and 43 days prior to RNA extraction. Extracted RNA can be stored at -80°C for at least 2 years.

[0077] MATERIALS / REAGENTS (Storage & Stability) & EQUIPMENT

[0078] A. Reagents and Equipment

[0079] B. Clean Room Equipment and Consumables

[0080] C. Post-PCR Sequencing Room

[0081] Validation of New Reagents

[0082] 1. New reagent lots and shipments are validated in parallel with existing reagents before being put into use.

[0083] 2. New reagents are aliquoted under the hood in the Clean Room / Main Lab.

[0084] 3. New reagents are run from RNA of a previously run sample and compared for concordance of results.

[0085] 4. For all reagents, a minimum of 5 patient samples and a Zyagen Human Total RNA are run with old and new lots. 5. An r of >0.95 and a p<0.05 are the acceptance criteria.

[0086] 6. Reagents and solution are labeled with: a. Lot number b. Content and quantity c. Date prepared / opened d. Expiration date

[0087] 7. Reagents from different lots are not mixed.

[0088] REAGENT AND SAMPLE PREPARATION

[0089] Isolating RNA from skin samples of unaffected and affected skin scrapings

[0090] NOTE: Work areas are cleaned daily with appropriate disinfectant prior to beginning the day’s work. All samples are labeled with their respective sample identifiers, using 2 identifiers when space is available.

[0091] Reagent Preparation

[0092] 1. Before first use of reagent kit. a. Prepare the Wash Solutions from the concentrates. i. Add 10 mL of 100% Isopropanol to Wash Solution 1 Concentrate, mix gently by inverting bottle back and forth and store at room temperature. ii. Add 48 mL 100% Ethanol to Wash Solution 2 Concentrate, mix gently by inverting bottle back and forth and store at room temperature.

[0093] Check box on each bottle, date, and initial that reagent preparation was performed.

[0094] PROCEDURE

[0095] Isolation of RNA from skin scrapings

[0096] Extract RNA with the modified tissue samples protocol.

[0097] 1 . Turn on KingFisher® Flex instrument.

[0098] 2. Decontaminate work bench and pipettes with 70% isopropanol before the start of reagent preparation.

[0099] 3. Prepare DNase solution and binding beads as indicated in Tables 1 and 2, respectively. a. Prepared DNase and binding beads mixes should be stored at 4°C until use.

[0100] Table 1 Total DNase Solution

[0101] Table 2

[0102] 4. Set up the processing plates shown in Table 3.

[0103] Table 3:

[0104] 5. Pipet the entire sample lysate volume of each clinical sample into a homogenizer (e.g. QIAshredder spin column) placed in a 2 mL collection tube. a. Include a separate sample containing 200 pl of extraction buffer as a negative control.

[0105] 6. Centrifuge the QIAshredder in a centrifuge for 2 minutes at maximum speed to homogenize the sample.

[0106] 7. Transfer flow through into a 96 Deep-well plate using a 4-channel pipette.

[0107] 8. Cover plate with an adhesive film and shake for 5 minutes at 1150 rpm on the microplate shaker.

[0108] 9. Add 200 pL of 100% isopropanol to each sample. Cover plate with an adhesive film and shake for 2 minutes at 950 rpm.

[0109] 10. Add 20 pL of the prepared nucleic acid binding beads mix to each sample and shake for 5 minutes at 950 rpm. a. Vortex binding bead mix for 1 minute at high speed prior to adding to samples.

[0110] 11 . Open Bindit 4.0 KingFisher® Flex software.

[0111] 12. Connect computer to KingFisher®.

[0112] 13. Select program.

[0113] 14. Start the run and load the prepared sample and processing plates in their positions when prompted by the instrument (see Table 4).

[0114] 15. Set timer for 30 minutes for addition of rebinding buffer and isopropanol. 16. When the KingFisher® prompts: a. Remove the DNase plate from the KingFisher®. b. Add 50 pL of rebinding buffer to each well. c. Add 100 pL 100% isopropanol to each well.

[0115] DO NOT PRE-MIX the Rebinding Buffer and isopropanol.

[0116] 17. Load the DNase plate back onto instrument and push Start.

[0117] 18. At the end of the run, remove all plates as prompted by the KingFisher®.

[0118] 19. Quantitate RNA using NanoDrop 8000. a. Alternatively, or in addition to NanoDrop, quantitate using HS RNA Qubit kit according to the manufacturer’s instructions.

[0119] 20. Transfer specimens to pre-labeled 1.5 mL centrifuge tubes and store at -80°C.

[0120] NOTE: Optional step - if the eluate is observed to be pigmented or contains excess bead residue after the final elution step, the sample can be purified to remove melanin and other contaminants including excess binding beads from the RNA.

[0121] Optional - PCR Inhibitor Removal Protocol

[0122] 1 . Insert column into a collection tube.

[0123] 2. Open the cap and add 600 pL of Prep-Solution.

[0124] 3. Centrifuge at 8,000 x g for 3 minutes.

[0125] 4. Transfer the prepared column to a clean, labeled 1.5 mL microcentrifuge tube and discard the collection tube with the flow through.

[0126] 5. Transfer the RNA to be purified onto the prepared column.

[0127] 6. Centrifuge at 8,000 x g for 1 minute.

[0128] 7. Store RNA at -80°C.

[0129] Reverse Transcription (RT) of RNA to cDNA

[0130] 1) Add the following reverse transcription reagents to each sample tube or 96 well PCR plate. a. Include the following controls: i. Negative control (8 pl KingFisher® extraction control) ii. Positive control (50 ng Zyagen high-quality total RNA)

[0131] 2) If using a 96 well PCR plate, seal with an adhesive film. Vortex samples to mix and centrifuge briefly to collect contents. 3) Run thermocycler RNAseq RT protocol. Thermocycler conditions are as follows:

[0132] 1Samples may be held overnight at 4 °C or optionally stored at -20 °C for longer periods

[0133] Amplify Targets

[0134] 1) Prepare master mix of the following reagents. Calculate volume with 10% overage to account for pipetting errors.

[0135] 2) Gently vortex master mix and centrifuge briefly to collect contents.

[0136] 3) Add 10 pl of master mix to each RT sample (20 pl final volume). Seal 96 well plate (if using) with new adhesive film. Vortex to mix and centrifuge to collect contents.

[0137] 4) Load samples in thermocycler and run the protocol as follows a.

[0138] 1Samples can be held at 4 °C overnight or optionally stored at -20 °C for longer periods.

[0139] Partially Digest Primer Sequences

[0140] 1) T ransfer PCR tubes or 96 well PCR plate to the post-PCR sequencing room.

[0141] 2) Briefly vortex and centrifuge samples to collect contents.

[0142] 3) Add 2 pl of FuPa reagent to each sample to partially digest the primer sequences. a. Pipette slowly, as the FuPa reagent is viscous.

[0143] 4) Vortex to mix and centrifuge to collect contents.

[0144] 5) Load samples in the thermocycler and run the fupa program. 1Do not freeze samples after the run and proceed to ligation within 1 hour of completion.

[0145] Ligate Barcodes to Amplicons

[0146] Dilute stock barcodes (if needed)

[0147] 1) Thaw stock barcodes at room temperature and prepare dilutions for each barcode based on the following table.

[0148] Store diluted barcodes at -20 °C

[0149] Perform barcode ligation

[0150] 1) Add components below to each sample in the order listed in the table below.

[0151] 2) Vortex to mix and centrifuge to collect contents.

[0152] 3) Load samples in a thermocycler and run the RNAseq ligate program.

[0153] 1Proceed to library clean up within 1 hr or store at -20 °C for long-term storage.

[0154] Purify unamplified libraries

[0155] • Equilibrate clean-up beads to room temperature at least 30 minutes prior to library purification. Vortex for 1 minute to thoroughly resuspend beads immediately before use.

[0156] • Use freshly prepared 70% ethanol (EtOH). Calculate for 300 pl 70% EtOH per sample plus 15% overage.

[0157] 1) Transfer libraries to a 96 well Midi plate.

[0158] 2) Add 45 pl of clean-up beads per sample. Pipette up and down 5X to mix. Try to avoid generating excessive frothing / bubbles.

[0159] 3) Incubate for 5 minutes.

[0160] 4) Place plate on a 96 magnetic rack. Incubate for 2 minutes or until solution clears and magnetic beads are thoroughly adhered to the well walls. 5) Carefully aspirate and discard solution.

[0161] 6) Add 150 l of 70% EtOH to each sample. Move the place diagonally down and to the right to the next series of magnets. Allow beads to pellet to the opposite side of the well wall. Return the plate to the original position and allow the beads to move to the original position.

[0162] 7) Aspirate and discard solution without disturbing pellet.

[0163] 8) Repeat steps 6 & 7 for a second wash.

[0164] 9) Keep the plate on the magnet and use a multi-channel pipettor to remove residual EtOH. Air-dry beads for 3-5 minutes.

[0165] 10) Remove the plate from the magnet and add 45 pl of Low TE per sample. Pipette up and down 10X or until the beads are evenly dispersed.

[0166] 1 1) Incubate resuspended samples for 5 minutes at room temperature.

[0167] 12) Place plate on the magnet and incubate for 2 minutes or until the solution has cleared.

[0168] 13) T ransfer 45 pl of purified library to a new PCR tube.

[0169] Quantify libraries by qPCR

[0170] Serially dilute E. coli DH10B Ion Control Libraries (if needed)

[0171] 1) Prepare 3 10-fold serial dilutions of E. coli DH10B control libraries, starting with the stock 68 pM tube. a. Add 10 pl of stock (68 pM) E. coli DH1 OB library into 90 pl NF-H2O (6.8 pM final concentration) b. Repeat serial dilution twice to generate 0.68 pM and 0.068 pM dilutions. c. Include a NF-H2O negative control.

[0172] Prepare qPCR Master Mix

[0173] 1) Open the FASTionqpcr thermocycler program. Enter the run name and upload a template containing the sample layout or manually enter sample names in duplicate in the Plate tab.

[0174] 2) Prepare qPCR master mix for nsampies + 4stds + 2error according to the table below.

[0175] 3) Vortex master mix and centrifuge to collect contents. Aliquot 1 1 pl per well in an optical 96-well plate.

[0176] 4) Dilute RNA-seq NGS libraries 1 :100 in NF-H2O (2 pl library + 198 pl NF-H2O). Vortex for 5 seconds and centrifuge to collect contents. 5) Add 9 pl of diluted NGS libraries to each well. Include each sample in duplicate.

[0177] 6) Seal the 96-well plate with optical film. Vortex plate to mix and spin in a 96-well plate mini centrifuge to collect contents.

[0178] 7) Load the 96-well plate in the qPCR thermal cycler.

[0179] 8) Run the thermal cycler program with updated well layout, (approximately 35 minutes run time)

[0180] 9) Analyze and export the data and determine the undiluted library concentrations (pM).

[0181] Dilute RNA-seq NGS libraries and generate pooled amplicon libraries

[0182] 1) Dilute RNA-seq NGS libraries in NF-H2O to 50 pM, if possible (i.e., undiluted libraries > 50 pM). Dilutions will be based on the library prep dilution calculation sheet.

[0183] 2) For each sequencing chip, pool 3 pl of each library into a LoBind Eppendorf 1 .5 ml microcentrifuge tube. a. Add 4-5 pl of undiluted NGS libraries < 50 pM.

[0184] 3) Vortex for 5 sec and centrifuge to collect contents. Store pooled amplicon libraries at 4 °C until the Ion Chef™ and S5 Prime reagents are thawed and ready for setup.

[0185] Setup the Planned Runs in the Ion Reporter

[0186] Set up the Ion Chef™ and Initialize the S5 Prime Sequencer

[0187] 1) Thaw the Ion 550 Chef™ Reagents and S5 Prime Sequencing Reagents at least 45 min prior to setup and initialization.

[0188] 2) Load reagents and consumables in the Ion Chef™. Select Step-By-Step program for detailed instructions and see FIG.1 for Ion Chef™ schematic. a. Shake the Ion Chef™ 550 Solutions and Ion Chef™ 550 Reagents to mix contents. Lightly tap bottom of cartridges on the bench counter to collect contents.

[0189] 3) When all reagents and consumables have been loaded into the Ion Chef™, add 25 pl of each pooled amplicon library to the proper tubes of the Ion 550 Reagents cartridge (see FIG.2).

[0190] Ion Chef™ 550 Reagents cartridge: Positions 1 & 2 correspond to the first and second chips, respectively, to be sequenced.

[0191] 4) Close Ion Chef™ door until it locks into place. Proceed with the deck scan (~10 minutes).

[0192] 5) Verify the planned run names for each chip.

[0193] 6) Select the Ion Chef™ finish time for the following day. a. The templating run must end no sooner than 16 hours after initialization. 7) On the GeneStudio™ S5 prime home screen, select Initialize. Follow the prompts for removal and addition of reagents and consumables.

[0194] 8) Start the initialization process. a. Can be performed the evening before the sequencing run. Initializations must be repeated with new reagents and consumables if the sequencing run is not performed within 24 hours of initialization.

[0195] All references cited in this application are expressly incorporated by reference herein.

Claims

WHAT IS CLAIMED IS:Claim 1. A method comprising:(a) obtaining a skin sample;(b) extracting nucleic acid from the sample, wherein extracting comprises: i) lysing the skin sample using a homogenizer to generate a homogenized sample; ii) mixing the homogenized sample for about 5 minutes at about 1150 rpm; iii) adding isopropanol to each mixed homogenized sample and mixing for about an additional 2 minutes at about 950 rpm; iv) combining the mixed homogenized sample comprising isopropanol with beads comprising a nucleic acid binding surface and incubating for about 5 minutes at about 950 rpm to bind the nucleic acid to the beads; v) washing the beads and treating the washed beads with a DNase; vi) washing the DNase treated beads; and vii) eluting the nucleic acid from the beads to generate an isolated nucleic acid sample.Claim 2. The method of claim 1 , wherein the skin sample is stored in an extraction buffer for up to about 96 hours at room temperature.Claim 3. The method of either claim 1 or claim 2, wherein the method further comprises quantitating the isolated nucleic acid sample.Claim 4. The method of any one of claims 1-3, wherein the nucleic acid comprises DNA, RNA, or both DNA and RNA.Claim 5. The method of any one of claims 1-4, wherein the nucleic acid comprises RNA.Claim 6. The method of claim 5, wherein the method further comprises reverse transcription of RNA to cDNA.Claim 7. The method of any one of claims 1-6, wherein the isolated nucleic acid sample is treated to remove contaminants.Claim 8. The method of claim 5, wherein reverse transcription comprises incubating at about 25°C for about 10 minutes, then at about 42°C for about 60 minutes, then at about 85°C for about 5 minutes, and finally at about 4°C for up to about 18 hours, or optionally at - 20°C for up to one year.Claim 9. The method of any one of claims 1-8 further comprising amplifying a population of targets from the isolated nucleic acid sample to produce a population of amplified targets.Claim 10. The method of claim 9 further comprising ligating barcodes to the population of amplified targets, and sequencing the population of amplified targets.Claim 11. The method of any one of claims 1-10, wherein the skin sample is obtained by a non-invasive method.Claim 12. The method of claim 11 , wherein the skin sample is obtained by a curette.Claim 13. The method of any one of claims 1-10, wherein the skin sample is a FFPE sample.Claim 14. The method of any one of claims 2-13, wherein the extraction buffer contains beta-mercaptoethanol and / or Triton.Claim 15. The method of any one of claims 2-14, wherein the extraction buffer does not contain thiocyanic acid.Claim 16 The method of any one of claims 2-15, wherein the sample is stored in the extraction buffer at a temperature that is at least about -80°C for up to about 45 days.Claim 17. A kit comprising reagents and instructions for performing the method of any one of claims 1-16.

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