Reagent, kit and extraction method for extracting small RNA in circulating body fluid and library building method for small RNA in circulating body fluid

Abstract

The invention provides an extraction reagent for extracting small RNA (Ribonucleic Acid) in circulating body fluid. The extraction reagent comprises a splitting combination solution containing Tris-HCl, SDS (Sodium Dodecyl Sulfate), EDTA (Ethylene Diamine Tetraacetic Acid), NaCl, sodium citrate and guanidine isothiocyanate, the invention also provides a kit containing the extraction reagent, an extraction method and a library building method of small RNA in body fluid. According to the method, a set of special extraction process is established for extracting extremely trace small RNA in body fluid, and direct and efficient extraction of small RNA in circulating body fluid is realized by optimizing the components and the proportion of the cracking combination liquid and combining a conventional magnetic bead purification technology; in the library construction process, high-quality on-machine sequencing of the final library is realized by finely adjusting the proportion of the linker addition amount in each step and optimizing library extraction. According to the high-throughput sequencing method disclosed by the invention, the initial dosage of the sample is reduced while the operation process is simplified, and the success rate of library construction and the overall quality of the sequencing library are remarkably improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to extraction reagents, kits, extraction methods, and library construction methods for small RNAs in circulating body fluids. Background Technology

[0002] Small non-coding RNAs (sRNAs) refer to a class of non-coding RNA molecules, generally less than 200 nt in length, that exist in the body fluid environment. They mainly include miRNAs, tsRNAs (tRNA-derived small RNAs), rsRNAs (rRNA-derived small RNAs), and ysRNAs. Based on their origin and generation mechanism, these small RNAs can be further classified. For example, miRNAs originate from the processing of precursor miRNAs, while tsRNAs and rsRNAs are produced by cleavage at specific sites on tRNAs and rRNAs, respectively.

[0003] Circulating small non-coding RNAs (sRNAs) are a class of non-coding nucleic acid molecules that have attracted much attention in recent years. Their earliest discovery can be traced back to miRNAs in the blood. Most of these circulating sRNAs exist stably in free form or encapsulated in exosomes or protein complexes, thus resisting degradation by external RNases and exhibiting extremely high stability and cross-tissue propagation capabilities. With further research, numerous studies have revealed that certain non-coding small RNAs are stably present in various circulating body fluids such as serum, plasma, urine, and breast milk, and have significant physiological and pathological relevance.

[0004] During disease development and progression, the expression profiles of circulating small RNAs undergo significant changes. For example, in colorectal cancer, the expression of miR-19a and miR-22 is upregulated and downregulated, respectively. This imbalance in expression can affect tumor proliferation, migration, and metastasis by regulating their target genes (such as the tumor suppressor gene TIA1 and the oncogene HuR), indicating that circulating miRNAs play a crucial regulatory role in tumor development and progression. Furthermore, circulating miRNAs also act as epigenetic information carriers in the transgenerational genetic regulation of neuropsychiatric disorders such as depression, influencing offspring behavior and neural development by regulating embryonic DNA methylation reprogramming.

[0005] Because circulating non-coding small RNAs (cRNAs) are stably present in body fluids and exhibit tissue-specificity and correlation with pathological states, they have broad research and application value in clinical practice. Firstly, as biomarkers, they demonstrate superiority in the early diagnosis and prognostic assessment of diseases such as cancer. By integrating high-throughput sequencing with machine learning models (such as random forests), highly specific and accurate combinations of small RNAs can be screened from body fluids such as serum for non-invasive cancer detection. Secondly, some cRNAs also exhibit potential therapeutic targets; for example, tsRNAs with anti-tumor effects can exert therapeutic potential by inducing apoptosis or inhibiting tumor growth. Therefore, the development of sequencing technologies targeting cRNAs is particularly important in scientific research and clinical applications.

[0006] The basic workflow for circulating small RNA sequencing used in current research is as follows: extraction of total RNA – library construction – sorting of small RNA libraries – sequencing. First, total RNA is extracted from circulating body fluids using methods such as TRIzol method / silica membrane centrifugation adsorption column extraction method / magnetic bead separation extraction method, and then library construction is performed. The general workflow for library construction is as follows: (1) ligating 3' adapters, adding reverse transcription primers to block 3' adapters, and ligating 5' adapters in sequence; (2) performing reverse transcription of RNA using reverse transcription primers previously used to block 3' adapters; (3) introducing index primers containing NGS sequencing adapters and universal primers for PCR library amplification. Next, fragment sorting is performed on the constructed total RNA library to obtain small RNA libraries. The library sorting methods commonly used in the research are: (1) gel electrophoresis on non-denaturing PAGE gel, followed by gel cutting and recovery of the corresponding bands of small RNA libraries; (2) double-round magnetic bead screening using different amounts of purification magnetic beads. Finally, the small RNA library was subjected to NGS high-throughput sequencing to obtain the small RNA sequence and abundance information in circulating body fluids.

[0007] However, existing methods for constructing libraries using small RNAs from circulating body fluids have several shortcomings: ① Due to the low concentration of small RNAs in circulating body fluids, the ligation of adapters exhibits bias during the extraction, addition of 3' / 5' adapters, reverse transcription, and library amplification processes. With a fixed number of adapters in the RNA solution, adapters preferentially bind to long-chain RNAs with higher concentrations, thus significantly reducing the reverse transcription and amplification efficiency of small RNAs in circulating body fluids. This results in the inability to sort out small RNA libraries in subsequent library sorting operations, leading to a low success rate. ② The experimental procedures are cumbersome, requiring purification of PCR products, melting gel recovery after library sorting, or dual-wheel magnetic bead screening, leading to instability in library results. It is difficult to verify the same trend when performing low-throughput RT-qPCR validation.

[0008] In summary, there is a need to develop a method for high-throughput sequencing of circulating small RNAs that is easy to operate, has a high success rate, good reproducibility, and is suitable for large sample sizes. Summary of the Invention

[0009] To address the aforementioned technical limitations, this application proposes extraction reagents, kits, extraction methods, and library construction methods for small RNAs in circulating body fluids; these overcome the deficiencies and defects mentioned in the background art.

[0010] To achieve the above objectives, this application adopts the following technical solution: The inventive point of this application is to provide an extraction reagent for extracting small RNA from circulating body fluids. The small RNA extraction reagent includes a lysis binding solution comprising Tris-HCl at a final concentration of 60-80 mmol / L, SDS at a mass / volume percentage of 1-3%, EDTA at a concentration of 1-3 mmol / L, NaCl at a concentration of 130-150 mmol / L, sodium citrate at a concentration of 30-40 mmol / L, and guanidine isothiocyanate at a concentration of 1-3 mol / L.

[0011] Optionally, the small RNA extraction reagent described above further includes washing solution 1 and washing solution 2; wherein, washing solution 1 is prepared by mixing the lysis binding solution and anhydrous ethanol, and the content of anhydrous ethanol in washing solution 1 is 60%-75% by volume percentage; washing solution 2 is prepared by mixing ddH2O and anhydrous ethanol at a volume ratio of 1:(3-5), preferably 1:4.

[0012] Optionally, the circulating body fluids in the above-mentioned small RNA extraction reagent include blood, serum, plasma, urine, exosomes, or semen.

[0013] The second inventive point of this application is to provide an extraction kit for extracting small RNA from circulating body fluids, the small RNA extraction kit comprising the small RNA extraction reagents shown above.

[0014] The third inventive point of this application is to provide the application of the above-mentioned small RNA extraction reagent or the above-mentioned small RNA extraction kit in the extraction of small RNA from circulating body fluids.

[0015] The fourth inventive point of this application is to provide a method for extracting small RNA from circulating body fluids, comprising the following steps: 1) Vortex mix the circulating body fluid sample with the above-mentioned lysis binding solution; 2) Add proteinase K solution and vortex to mix; 3) Add the purified magnetic beads and vortex mix well; 4) Constant temperature incubation; 5) After incubation, add anhydrous ethanol, vortex to mix, let stand until clear, and then discard the supernatant. 6) Add the rinse solution 1 and mix well. Centrifuge briefly, let stand until clear, and then discard the supernatant. 7) Repeat the addition of the rinse solution 1, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 8) Add the rinse solution 2 and mix well. Centrifuge briefly, let stand until clear, and then discard the supernatant. 9) Repeat the addition of the rinsing solution 2, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 10) Add anhydrous ethanol, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 11) Centrifuge briefly and remove residual liquid; 12) After drying in a metal bath, add ddH2O and mix well, then incubate in a metal bath. 13) After instantaneous centrifugation, allow to stand until clear; 14) The supernatant is the small RNA extracted from the circulating body fluid.

[0016] Optionally, the above-described method for extracting small RNAs from circulating body fluids, The ratio of the circulating body fluid sample, lysis binding buffer, proteinase K solution, purified magnetic beads, and anhydrous ethanol was 1:(1.8-2.2):(0.08-0.12):(0.1-0.2):(4.8-5.9) by volume. The mixing time for steps 1) and 2) is 5-10 seconds, preferably 5 seconds; The mixing time for steps 3), 5), 6), 7), 8), 9), 10), and 12) is 10-15 seconds, preferably 15 seconds; In step 4), the incubation is carried out by water bath incubation at 60-65℃ or constant temperature forced air drying incubation, preferably at 60℃; the incubation time is 10-15 minutes, preferably 10 minutes; In steps 6) and 7), the amount of rinsing solution 1 added is 0.8-1.2 ml / time, preferably 1 ml; In steps 8) and 9), the amount of rinsing solution 2 added is 0.8-1.2 ml / time, preferably 1 ml; In steps 5)-10), the settling time is 2-5 minutes; In step 12), the temperature for drying and incubating the metal bath is 60℃-65℃, preferably 65℃, the amount of eluent added is 10-30μL, preferably 15μL, and the mixture is stirred at 1500rpm for 15-30 seconds, preferably 15 seconds. The incubation time in the metal bath is 5-10 minutes, preferably 5 minutes. In step 13), the settling time is 2-5 minutes.

[0017] The fifth inventive point of this application is to provide a method for constructing a library of small RNAs in circulating body fluids, comprising the following steps: a) Small RNAs in circulating body fluids are extracted using the extraction method of claim 6 or 7; b) Linking 3' adapters to small RNAs in circulating body fluids; c) Seal off any excess 3' connector; d) Ligate 5' adapters to small RNAs in circulating body fluids; e) Reverse transcription of small RNAs in circulating body fluids into cDNA; f) After PCR amplification and purification of the PCR products, sequencing libraries of small RNAs in circulating body fluids were obtained by enrichment.

[0018] Optionally, in the above library construction method, the dilution ratio of the 3' adapter, reverse transcription primer and 5' adapter is (adapter / primer):ddH2O=1:(15-20) by volume, preferably 1:19.

[0019] Optionally, in the above library construction method, during the library enrichment in step f), the amount of universal primers and index primers added is 0.5-1 μL, preferably 0.5 μL, and the PCR amplification program is 20-25 cycles, preferably 25 cycles.

[0020] Existing commercial small RNA library construction kits, such as NEB's NEBNext Multiplex Small RNA Library Prep Set and Novizan's VAHTS Small RNA Library Prep Kit for Illumina V2, generally employ a "library construction – small RNA library sorting – sequencing" approach for small RNA library construction. The specific library construction process is as follows: ① Extract total RNA from the sample; ② Denature 3' adapters and total RNA; ③ Ligate 3' adapters; ④ Block excess 3' adapters; ⑤ Ligate denatured 5' adapters; ⑥ cDNA synthesis; ⑦ Library PCR amplification; ⑧ Library purification and sorting; ⑨ Sequencing. Total RNA extraction can be performed using the TRIzol method, magnetic bead separation extraction, or silica gel membrane centrifugation column extraction. Library sorting can be performed using non-denaturing 6% PAGE gel electrophoresis to sort fragments by size, 3% agarose gel electrophoresis and a fully automated nucleic acid electrophoresis and gel recovery system to recover small RNA libraries of appropriate sizes, or using a dual-wheel magnetic bead method.

[0021] In addition, existing small RNA extraction kits, such as QIAGEN's miRNeasy Serum / Plasma Kit, use phenol / guanidine thiocyanate lysis combined with silica membrane purification technology. By adjusting the ethanol concentration and thus the binding conditions, small RNAs are specifically bound to the silica membrane. After small RNA extraction using the QIAGQEN kit, library construction can be performed directly, and the library results are as follows: Figure 1 As shown in the image. The quality control results indicate that fragments outside the small RNA library length range still exist in the final library, suggesting that long RNA fragments still exist in the RNA samples extracted using this small RNA extraction kit. If library sorting is not performed subsequently, it will still result in a low output of effective sequencing data.

[0022] Therefore, most existing methods are designed for extraction and library construction of samples with high abundance. However, small RNAs in body fluids are present in extremely small quantities, leading to high library construction failure rates, large sample volumes, and the inability to obtain consistent results from subsequent low-throughput RT-qPCR. To address these issues, improve the success rate of small RNA library construction in body fluids, reduce sample volumes to conventional levels, and ensure consistency between low-throughput validation results and high-throughput sequencing results, the applicant has developed a complete workflow.

[0023] First, this application establishes a proprietary extraction process for extremely small amounts of small RNA (<90 nt) in bodily fluids. This patent achieves direct and efficient extraction of small RNA from circulating bodily fluids by optimizing the components and ratios of the lysis binding solution and combining it with conventional magnetic bead purification technology.

[0024] Secondly, this application establishes an operational workflow to improve the quality and concentration of small RNA libraries. During library construction, this application achieves high-quality sequencing of the final library without the need for additional library sorting steps by finely adjusting the proportion of adapter addition and optimizing library extraction in each step. This application also provides a high-throughput sequencing method suitable for small RNA extraction and library construction from circulating body fluids. Compared to existing technologies, this method significantly simplifies the operational process, reduces the initial sample volume, and significantly improves the success rate of library construction and the overall quality of the sequencing library.

[0025] Specifically, the technical protection points intended in this application are as follows: (1) This application relates to a formulation of a lysis binding buffer for extracting small RNA, wherein the lysis binding buffer contains 60-80 mM Tris-HCl (pH 7.5) at a final concentration, 1-3% (wt / vol) SDS, 1-3 mM EDTA, 130-150 mM NaCl, 30-40 mM sodium citrate, and 1-3 M guanidine isothiocyanate; (2) This application provides a kit for extracting small RNA from circulating body fluids. The wash buffer 1 is a mixture of lysis binding buffer and anhydrous ethanol, wherein the ethanol content is 60%-75% (vol / vol). In this application, the proportion of wash buffer 1 can be adjusted according to different sample types; (3) The method for extracting small RNA using the kit in this application differs from the prior art in the following aspects: ① the proportions of circulating body fluid sample, lysis binding solution, proteinase K, purified magnetic beads and anhydrous ethanol added during magnetic bead binding; ② the elution volume, elution time and specific operating procedures during elution. (4) In the adapter ligation process of library construction in this application, the dilution ratio of 3' adapter, reverse transcription primer and 5' adapter is adapter / primer:ddH2O (vol:vol) = 1:19. At this dilution ratio, the influence of adapter contamination is minimized while ensuring that all small RNAs are ligated to adapters. Therefore, the dilution ratio of 3' adapter, reverse transcription primer and 5' adapter is an extremely important experimental detail. (5) In the library enrichment operation of the library construction in this application, the input amount of universal primers and index primers is 0.5 μL, and the PCR amplification program is 25 cycles. With this amount of adapter input and number of cycles, the contamination of adapters can be avoided to the greatest extent while ensuring that the library concentration reaches the sequencing concentration. Therefore, the input amount of amplification adapters and the setting of the PCR amplification program are extremely important experimental details in this step.

[0026] Compared with the prior art, this application has the following advantages: (1) Database construction results using the NEB method: In this patent, total RNA was extracted using the NEB RNA Extraction Kit, and a Small RNA Library Construction Kit was used as a control for library construction. First, total RNA was extracted from the sample using the NEB Magnetic Bead Extraction / Silica Membrane Centrifugal Adsorption Column Extraction Kit. The serum sample volume was 3 mL, and RNA was eluted with 30 μL of elution buffer. Subsequently, a small RNA library was constructed using the NEB Small RNA Diversity Library Construction Kit. Purification and recovery of the PCR products were performed using the NEB Silica Membrane Centrifugal Adsorption Column Extraction PCR & DNA Purification Kit, and the library products were eluted with 25 μL of elution buffer. After library construction, the small RNA library was separated and screened by electrophoresis using a 6% non-denaturing polyacrylamide gel (PAGE). The DNA Marker, 6× loading buffer, and recommended electrophoresis conditions were followed according to the kit instructions. The gel was stained with Gel Red dye and observed under a gel imaging system. Figure 2As shown, no obvious small RNA library-specific bands were observed. Subsequently, following the manufacturer's instructions, the corresponding band regions of the small RNA library were recovered from the gel by gel excision, and the recovered products were further purified by alcohol precipitation. Finally, the library concentration was quantified using a Qubit fluorometer, and the results showed that the library concentration was low. The quality control results of the small RNA library obtained using the NEB kit are shown below. Figure 3 As shown, five experiments were conducted, and the library was successfully constructed twice, with an overall experimental repeatability rate of less than 40%.

[0027] (2) Small RNA library construction results in this patent: ① The quality control test results of the small RNA extracted using this method ( Figure 4 The results showed that the RNA fragments were mostly less than 200 nt, with minimal contamination from long fragments, meeting the requirements for small RNA library construction. This is consistent with the quality control results of the total RNA library. Figure 5 In contrast, the electrophoretic pattern corresponding to total RNA library construction shows broad peaks, while the RNA fragments extracted by this method are concentrated in the corresponding small fragment range. Figure 6 The presence of virtually no large RNA fragments indicates that this extraction method is specific for extracting small RNAs.

[0028] ② The small RNA libraries constructed using this method in circulating body fluids (serum, plasma, urine, seminal plasma) exhibited obvious characteristic peaks in quality control testing. Figure 6-9 This further supports the preservation effect and library construction efficiency of the method of this patent on small RNA.

[0029] ③ After constructing a small RNA library using serum samples, the library sequencing results were compared with the library constructed using the QIAGEN small RNA extraction kit and the library constructed using the NEB library construction kit. Figure 11 There was no significant difference in the distribution of small RNA species.

[0030] ④ A correlation analysis was performed to compare the results of small RNA libraries constructed using this method, libraries constructed using the QIAGEN small RNA extraction kit, and libraries constructed using the NEB library construction kit. Figure 12 As shown, the small RNA library obtained by this method has good correlation with the library obtained by traditional methods.

[0031] ⑤ Select miRNA markers based on sequencing results, and perform RT-qPCR verification on the small RNA library obtained using this method, such as... Figure 13 As shown in the figure, the verification results show good consistency. Attached Figure Description

[0032] Figure 1The image shown is a quality control diagram of a library constructed after small RNA extraction using the QIAGEN kit, as described in one embodiment of this application.

[0033] Figure 2 The image shown is a gel electrophoresis diagram of small RNA library sorting constructed using the NEB kit, as described in one embodiment of this application.

[0034] Figure 3 The image shows the quality control results of a small RNA library constructed using the NEB kit, as illustrated in one embodiment of this application.

[0035] Figure 4 The image shown is an embodiment of this application, illustrating the quality control results of small RNA extracted using the reagent kit of this patent.

[0036] Figure 5 The image shown is a quality control result of total RNA extraction after library construction using a commercially available serum / plasma RNA extraction kit, as described in one embodiment of this application.

[0037] Figure 6 The image shown is an embodiment of this application, illustrating the quality control results of a serum small RNA library constructed using the method described in this patented application.

[0038] Figure 7 The image shown is an embodiment of this application, illustrating the quality control results of a plasma small RNA library constructed using the method described in this patented application.

[0039] Figure 8 The image shown is an embodiment of this application, illustrating the quality control results of a urine small RNA library constructed using the method described in this patented application.

[0040] Figure 9 The image shown is an embodiment of this application, illustrating the quality control results of a seminal plasma small RNA library constructed using the method of this patent.

[0041] Figure 10 The image shown is an embodiment of this application, illustrating the quality control results of a small RNA library constructed using the method of this patent with gradient serum levels.

[0042] Figure 11 The pie chart shown here, as an embodiment of this application, illustrates the proportion of different types of small RNAs in the small RNA library constructed by the method of this patent and the small RNA library constructed by a commercially available kit.

[0043] Figure 12 The following is a correlation analysis of the small RNA library sequencing results (CH) obtained by this method and the small RNA library sequencing results obtained using commercially available patented magnetic beads (XH) in one embodiment of this application.

[0044] Figure 13 The illustration shows an RT-qPCR consistency analysis of a small RNA library obtained by the method of this patent in one embodiment of this application. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0046] Unless otherwise defined, 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0047] (a) This patent provides a lysis binding solution for extracting small RNAs from circulating body fluids.

[0048] Lysis binding buffer is suitable for extracting small RNAs from circulating body fluids (such as blood, serum, plasma, urine, exosomes, semen, etc.), and has the advantages of high extraction rate, high extraction purity, and suitability for a wide variety of samples.

[0049] The lysis binder contains 60-80 mM Tris-HCl (pH 7.5) at a final concentration, 1-3% (wt / vol) SDS, 1-3 mM EDTA, 130-150 mM NaCl, 30-40 mM sodium citrate, and 1-3 M guanidine isothiocyanate.

[0050] The materials required to prepare the lysis binding solution are: Tris-HCl, SDS, EDTA-2Na, NaCl, and sodium citrate.

[0051] (1) Prepare 1M Tris-HCl (pH 7.5): Weigh 6.055g Tris, add ddH2O to 40mL, adjust the pH to 7.5 with concentrated HCl, and add ddH2O to make up to 50mL; (2) Prepare 0.5M EDTA (pH 8.0): Weigh 9.305g EDTA-2Na, add ddH2O to 40mL, adjust pH to 8.0 with NaOH, and add ddH2O to make up to 50mL; (3) Prepare a 10% (wt / vol) SDS solution: Weigh 5g SDS, add ddH2O to 40mL, stir to dissolve and then make up to 50mL; (4) Prepare 5M NaCl solution: Weigh 14.61g NaCl, add ddH2O to 40mL, stir to dissolve and then make up to 50mL; (5) Prepare 1M sodium citrate solution: Weigh 2.16g sodium citrate, add ddH2O to 9mL, dissolve and then make up to 10mL; (6) Preparation of lysis binding solution: According to the lysis binding solution formula, use solid guanidine isothiocyanate powder and the above stock solution to prepare a final concentration of 60-70 mM (e.g., 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, etc.) Tris-HCl (pH 7.5), 1-3% (wt / vol) (e.g., 1%, 1.5%, 2%, 2.5%, 3%) SDS, 1-3 mM (1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM) EDTA, 130-150 mM (e.g., 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, etc.) NaCl, 30-40 mM (e.g., 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM). Sodium citrate (mM, 40 mM, etc.) and cleavage binders of guanidine isothiocyanate (1-3 M, e.g., 1 M, 1.5 M, 2 M, 2.5 M, 3 M) at 1-3 M (e.g., 1 M, 1.5 M, 2 M, 2.5 M, 3 M).

[0052] (ii) This patent also provides a kit for extracting small RNAs from circulating body fluids.

[0053] The kit includes wash buffer 1, wash buffer 2, purified magnetic beads, and the aforementioned lysis binding solution. The preparation methods for wash buffer 1 and wash buffer 2 are as follows: (1) Preparation of rinsing solution 1: Rinsing solution 1 is a mixture of lysis and binding solutions, in which the ethanol content is 60%-75% (vol / vol). Rinsing solution 1 is mixed and used for later use. Washing solution 1 should be prepared and used immediately. (2) Preparation of rinsing solution 2: Add ddH2O and anhydrous ethanol at a ratio of 1:4 (vol / vol), vortex mix well and set aside to form a final concentration of 80% (vol / vol) ethanol. Washing solution 2 should be prepared and used immediately.

[0054] (iii) This patent also provides an extraction method for extracting small RNA from circulating body fluids.

[0055] The specific procedure for extracting small RNA using this lysis binding buffer is as follows: Preheat the lysis binding solution in a 60°C water bath until the solution is clear and transparent; remove the purified magnetic beads from the 4°C refrigerator 30 minutes in advance and allow them to return to room temperature. (1) Add circulating body fluid sample and (1.8-2.2) times the sample volume of lysis binding solution to centrifuge tube, and vortex mix for 5 seconds; (2) Add (0.08-0.12) times the sample volume of proteinase K solution, and vortex again for 5 seconds; (3) Add (0.1-0.2) times the sample volume of purification magnetic beads and vortex thoroughly for 15 seconds; (4) Incubate in a 60℃ water bath or constant temperature drying oven for 10 minutes; (5) After incubation, remove the tube (if using a water bath, wipe the water droplets off the tube wall), add (4.8-5.9) times the volume of the sample in anhydrous ethanol, vortex mix for 15 seconds and place it on a magnetic rack. (6) After the solution has settled and become clear, use a pipette to remove the supernatant; (7) Add 1 mL of rinsing solution 1 to the centrifuge tube, mix well for 15 seconds, and then transfer all the liquid to a 1.5 mL EP tube; (8) After instantaneous centrifugation, place the tube on a 1.5 / 2.0 mL magnetic rack and let it stand for 2–5 minutes until it becomes clear, then discard the supernatant. (9) Add 1 mL of washing solution 1, mix well for 15 seconds, centrifuge briefly, place on a magnetic rack again to clarify for 2–5 minutes, and discard the supernatant; (10) Add 1 mL of rinsing solution 2, mix well for 15 seconds, centrifuge briefly, place on a magnetic rack and let stand for 2–5 minutes, then discard the supernatant; (11) Repeat step (10) once; (12) Add 1 mL of anhydrous ethanol, mix well for 15 seconds, centrifuge briefly, place in a magnetic rack again and let stand for 2–5 minutes, then discard the supernatant. (13) After instantaneous centrifugation, continue to place the liquid in a magnetic rack and use a 100μL or 200μL pipette to completely remove any residual liquid; (14) Open the centrifuge tube cap, place it in a metal bath at 60 ℃-65 ℃ to dry until the magnetic beads have no metallic luster, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate in a metal bath at 60 ℃-65 ℃ for 5 minutes. (15) Centrifuge briefly to concentrate the liquid to the bottom of the tube, and place it in a magnetic rack to stand for 2–5 minutes until it becomes clear; (16) Carefully transfer the supernatant to a new centrifuge tube, avoiding the introduction of magnetic beads. It can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0056] (iv) This patent also provides an optimized small RNA library construction process: The small RNA library construction procedure in this experiment is based on a commercially available small RNA library construction kit.

[0057] The 3' adapter, reverse transcription primer, and 5' adapter were all diluted at a ratio of adapter / primer:ddH2O (vol:vol) = 1:19.

[0058] (1) 3' connector connection: Remove the 3' adapter, 3' adapter buffer, and 3' adapter enzyme mix, thaw and mix them, briefly centrifuge to collect the contents to the bottom of the tube, and place them on ice for later use. All subsequent steps shall be performed on ice.

[0059] ① Template and 3' adapter denaturation: Prepare the reaction system in RNase-free PCR tubes according to Table 1 below.

[0060] Table 1

[0061] ② Place the PCR tube in a PCR instrument preheated to 70°C for 2 min and react for 2 min. After the reaction is complete, immediately remove the tube and place it on ice for 2 min.

[0062] ③ Add the components shown in Table 2 to the reaction tube in step ① in sequence: Table 2

[0063] ④ Gently tap the bottom of the tube to mix the components thoroughly, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Place the reaction tube in a PCR instrument with a heated lid and run the program shown in Table 3 below: Table 3

[0064] (2) Seal the extra 3' joint ① Prepare the reaction system as shown in Table 4 below: Table 4

[0065] ② Gently tap the bottom of the tube to thoroughly mix the components, then briefly centrifuge to collect the mixture at the bottom. Place the tube in a PCR instrument with a heated lid and run the program shown in Table 5 below: Table 5

[0066] (3) 5' connector connection Remove the 5' connector, 5' connector buffer, and 5' connector enzyme mix, thaw and mix them, briefly centrifuge to collect them to the bottom of the tube, and place them on ice for later use. All the following steps shall be performed on ice.

[0067] ① 5' adapter denaturation: Place the diluted 5' adapter in a PCR instrument at 70°C for 2 min, and immediately remove it and place it on ice after the reaction is complete.

[0068] ② Prepare the 5' connector reaction system according to Table 6 below: Table 6

[0069] ③ Gently tap the bottom of the tube to thoroughly mix the components, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Place the reaction tube in a PCR instrument with a heated lid and run the program shown in Table 7 below: Table 7

[0070] (4) cDNA synthesis Remove the reverse transcription buffer and reverse transcription enzyme mix, thaw and mix them, briefly centrifuge to collect them to the bottom of the tube, and place them on ice for later use.

[0071] ① Prepare the reverse transcription reaction system according to Table 8 below: Table 8

[0072] ② Gently tap the bottom of the tube to thoroughly mix the components, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Place the reaction tube in a PCR instrument with a heated lid and run the program shown in Table 9 below: Table 9

[0073] (5) Library enrichment Take out the universal primers, index primers and PCR amplification solution mix, thaw and mix well, then place on ice for later use.

[0074] ① Prepare the reaction system according to Table 10 below: Table 10

[0075] ② Mix the above reaction solution thoroughly, briefly centrifuge to collect the residue at the bottom of the tube. Place the reaction tube in a PCR instrument with a heated lid and run the program shown in Table 11 below: Table 11

[0076] (6) Purification of PCR products ①Take the magnetic bead solution out of the 4°C refrigerator 30 minutes in advance and let it stand to allow its temperature to equalize to room temperature.

[0077] ② Invert or vortex to mix the magnetic bead solution thoroughly. Add 50 μL of the magnetic bead solution to the DNA sample and gently pipette 10 times to mix thoroughly.

[0078] ③Incubate at room temperature for 10 min to allow DNA to bind to the magnetic beads.

[0079] ④ Place the sample on the magnetic rack and wait for the solution to clarify (about 5 minutes), then carefully remove the supernatant.

[0080] ⑤ Keep the sample on the magnetic rack at all times, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0081] ⑥ Repeat step 5 once, for a total of two rinses.

[0082] ⑦ Keep the sample on the magnetic rack at all times, and open the lid to dry the magnetic beads for about 5-10 minutes at room temperature.

[0083] ⑧ Remove the sample from the magnetic rack, add an appropriate amount of nuclease-free water, vortex or pipette to mix thoroughly, and let stand at room temperature for 2 minutes. Let stand on the magnetic rack for 5 minutes to allow the solution to clarify. ⑨ After cleaning, carefully aspirate the supernatant into a new nuclease-free centrifuge tube.

[0084] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0085] Example 1 1. The reagent preparation ratio in this example is: (1) Ligation binding solution: 75 mM Tris-HCl (pH 7.5), 1.5% (wt / vol) SDS, 1.5 mM EDTA, 150 mM NaCl, 37.5 mM sodium citrate, 1.5 M guanidine isothiocyanate; (2) Rinse solution 1: lysis binder: anhydrous ethanol ratio of 2:3 (vol / vol); (3) Rinse solution 2: ddH2O: anhydrous ethanol is 1:4 (vol / vol).

[0086] 2. Extract small RNA from serum using the above-mentioned lysis binding buffer. The steps include: (1) After mixing the serum sample, centrifuge it, add 2 times the sample volume of lysis binding buffer, and vortex to mix. (2) Add 0.1 times the sample volume of proteinase K and vortex to mix; (3) Add 0.15 times the sample volume of purified magnetic beads, vortex to mix, and incubate at 60°C for 10 min; (4) Add 5.9 times the volume of anhydrous ethanol, vortex mix, and discard the supernatant after the magnetic beads are adsorbed by the magnetic rack. (5) Add 1 mL of rinsing solution 1, vortex to mix, transfer all liquid to a new EP tube, and use a magnetic rack to remove the supernatant; (6) Add 1 mL of rinsing solution 2, mix well and centrifuge briefly. Use a magnetic rack to remove the supernatant. Repeat once, and wash twice with rinsing solution 2. (7) Add 1 mL of anhydrous ethanol, mix well and centrifuge briefly, then use a magnetic rack to adsorb and discard the supernatant. (8) After opening the centrifuge tube cap, place it in a 65°C metal bath to dry the magnetic beads, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate in a 65°C metal bath for 5 minutes. (9) Centrifuge briefly to concentrate the liquid to the bottom of the tube, then place it in a magnetic rack and let it stand until the solution becomes clear; (10) Transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0087] 3. Construction of small RNA library from serum samples: (1) Template denaturation: The RNA sample and the diluted 3' adapter were mixed and denatured at 70°C for 2 min, and then placed on ice; (2) 3' connector connection: Add 3' connector buffer and 3' connector enzyme mix to the product from the previous step, mix well, and incubate at 25°C for 1 hour; (3) Blocking extra 3' linkers with reverse transcription primers: Add diluted reverse transcription primers to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 75℃, 5 min; 37℃, 15 min; 25℃, 15 min; (4) 5' connector denaturation: The diluted 5' connector was denatured at 70°C for 2 min and then placed on ice; (5) 5' connector connection: Add 5' connector, 5' connector buffer and 5' connector Enzymemix to the product of step (3), mix well and incubate at 25°C for 1 h; (6) Reverse transcription: Add reverse transcription buffer and reverse transcription enzyme mix to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 50℃ for 1 h; 80℃ for 5 min; (7) PCR library amplification: Add PCR amplification mix, universal primers and index primers to the product from the previous step, mix well and place on a PCR instrument for reaction. The reaction program is 94℃ denaturation for 3 min; 94℃, 15 s, 65℃, 15 s, 72℃, 15 s, for a total of 22-25 cycles; 72℃, 1 min.

[0088] PCR products were purified using a silica gel membrane purification column to obtain a pure library. The FragmentAnalyzer quality control results are as follows: Figure 6 As shown.

[0089] Example 2 1. The reagent preparation ratio in this example is: (1) Ligation binding solution: 72 mM Tris-HCl (pH 7.5), 1.2% (wt / vol) SDS, 1 mM EDTA, 140 mM NaCl, 35 mM sodium citrate, 1.7 M guanidine isothiocyanate; (2) Rinse solution 1: lysis binder: anhydrous ethanol ratio of 1.8:3 (vol / vol); (3) Rinse solution 2: ddH2O: anhydrous ethanol is 1:4 (vol / vol).

[0090] 2. Extract small RNA from plasma using the above-mentioned lysis binding buffer. The steps include: (1) After mixing the plasma sample, centrifuge it, add 2.1 times the sample volume of lysis binding buffer, and vortex mix. (2) Add 0.08 times the sample volume of proteinase K and vortex to mix; (3) Add 0.15 times the sample volume of purified magnetic beads, vortex to mix, and incubate at 60°C for 10 min; (4) Add 5 times the volume of anhydrous ethanol, vortex mix, and discard the supernatant after the magnetic beads are adsorbed by the magnetic rack. (5) Add 1 mL of rinsing solution 1, vortex to mix, transfer all liquid to a new EP tube, and use a magnetic rack to adsorb and discard the supernatant; (6) Add 1 mL of rinsing solution 2, mix well and centrifuge briefly. Use a magnetic rack to remove the supernatant. Repeat once, and wash twice with rinsing solution 2. (7) Add 1 mL of anhydrous ethanol, mix well and centrifuge briefly, then use a magnetic rack to adsorb and discard the supernatant; (8) After opening the centrifuge tube cap, place it in a 65°C metal bath to dry the magnetic beads, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate at 65°C for 5 minutes. (9) Centrifuge briefly to concentrate the liquid to the bottom of the tube, then place it in a magnetic rack and let it stand until the solution becomes clear; (10) Transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0091] 3. Construction of small RNA library from plasma samples: (1) Template denaturation: The RNA sample and the diluted 3' adapter were mixed and denatured at 70°C for 2 min, and then placed on ice; (2) 3' connector connection: Add 3' connector buffer and 3' connector enzyme mix to the product from the previous step, mix well, and incubate at 25°C for 1 hour; (3) Blocking extra 3' linkers with reverse transcription primers: Add diluted reverse transcription primers to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 75℃, 5 min; 37℃, 15 min; 25℃, 15 min; (4) 5' connector denaturation: The diluted 5' connector was denatured at 70°C for 2 min and then placed on ice; (5) 5' connector connection: Add 5' connector, 5' connector buffer and 5' connector Enzymemix to the product of step (3), mix well and incubate at 25°C for 1 h; (6) Reverse transcription: Add reverse transcription buffer and reverse transcription enzyme mix to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 50℃ for 1 h; 80℃ for 5 min; (7) PCR library amplification: Add PCR amplification mix, universal primers and index primers to the product from the previous step, mix well and place on a PCR instrument for reaction. The reaction program is 94℃ denaturation for 3 min; 94℃, 15 s, 65℃, 15 s, 72℃, 15 s, for a total of 22-25 cycles; 72℃, 1 min.

[0092] PCR products were purified using a silica gel membrane purification column to obtain a pure library. The FragmentAnalyzer quality control results are as follows: Figure 7 As shown.

[0093] Example 3 1. The reagent preparation ratio in this example is: (1) Ligation binding solution: 80 mM Tris-HCl (pH 7.5), 1% (wt / vol) SDS, 3 mM EDTA, 150 mM NaCl, 40 mM sodium citrate, 1M guanidine isothiocyanate; (2) Rinse solution 1: lysis binding solution: anhydrous ethanol is 2:3 (vol / vol).

[0094] (3) Rinse solution 2: ddH2O: anhydrous ethanol ratio of 1:4 (vol / vol) 2. Extract small RNAs from urine using the above-mentioned lysis binding buffer. The steps include: (1) After mixing the urine sample, centrifuge it, add 2 times the sample volume of lysis binding buffer, and vortex mix. (2) Add 0.08 times the sample volume of proteinase K and vortex to mix; (3) Add 0.1 times the sample volume of purified magnetic beads, vortex to mix, and incubate at 60°C for 10 min; (4) Add 5.5 times the volume of anhydrous ethanol, vortex mix, and discard the supernatant after the magnetic beads are adsorbed by the magnetic rack. (5) Add 1 mL of rinsing solution 1, vortex to mix, transfer all liquid to a new EP tube, and use a magnetic rack to adsorb and discard the supernatant; (6) Add 1 mL of rinsing solution 2, mix well and centrifuge briefly. Use a magnetic rack to remove the supernatant. Repeat once, and wash twice with rinsing solution 2. (7) Add 1 mL of anhydrous ethanol, mix well and centrifuge briefly, then use a magnetic rack to adsorb and discard the supernatant; (8) After opening the centrifuge tube cap, place it in a 65°C metal bath to dry the magnetic beads, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate at 65°C for 5 minutes. (9) Centrifuge briefly to concentrate the liquid to the bottom of the tube, then place it in a magnetic rack and let it stand until the solution becomes clear; (10) Transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0095] 3. Construction of small RNA library from urine samples: (1) Template denaturation: The RNA sample and the diluted 3' adapter were mixed and denatured at 70°C for 2 min, and then placed on ice; (2) 3' connector connection: Add 3' connector buffer and 3' connector enzyme mix to the product from the previous step, mix well, and incubate at 25°C for 1 hour; (3) Blocking extra 3' linkers with reverse transcription primers: Add diluted reverse transcription primers to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 75℃, 5 min; 37℃, 15 min; 25℃, 15 min; (4) 5' connector denaturation: The diluted 5' connector was denatured at 70°C for 2 min and then placed on ice; (5) 5' connector connection: Add 5' connector, 5' connector buffer and 5' connector Enzymemix to the product of step (3), mix well and incubate at 25°C for 1 h; (6) Reverse transcription: Add reverse transcription buffer and reverse transcription enzyme mix to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 50℃ for 1 h; 80℃ for 5 min; (7) PCR library amplification: Add PCR amplification mix, universal primers and index primers to the product from the previous step, mix well and place on a PCR instrument for reaction. The reaction program is 94℃ denaturation for 3 min; 94℃, 15 s, 65℃, 15 s, 72℃, 15 s, for a total of 22-25 cycles; 72℃, 1 min.

[0096] PCR products were purified using a silica gel membrane purification column to obtain a pure library. The FragmentAnalyzer quality control results are as follows: Figure 8 As shown.

[0097] Example 4 1. The reagent preparation ratio in this example is: (1) Ligation binding solution: 80 mM Tris-HCl (pH 7.5), 3% (wt / vol) SDS, 3 mM EDTA, 150 mM NaCl, 35 mM sodium citrate, 3 M guanidine isothiocyanate; (2) Rinse solution 1: lysis binding solution: anhydrous ethanol is 1:3 (vol / vol).

[0098] (3) Rinse solution 2: ddH2O: anhydrous ethanol ratio of 1:4 (vol / vol) 2. Extract small RNA from seminal plasma using the above-mentioned lysis binding buffer. The steps include: (1) After mixing the seminal plasma sample, centrifuge it, add 2.2 times the sample volume of lysis binding solution, and vortex mix. (2) Add 0.12 times the sample volume of proteinase K and vortex to mix; (3) Add 0.2 times the sample volume of purified magnetic beads, vortex to mix, and incubate at 60°C for 10 min; (4) Add 4.8 times the volume of anhydrous ethanol, vortex to mix, and discard the supernatant after the magnetic beads are adsorbed by the magnetic rack. (5) Add 1 mL of rinsing solution 1, vortex to mix, transfer all liquid to a new EP tube, and use a magnetic rack to adsorb and discard the supernatant; (6) Add 1 mL of rinsing solution 2, mix well and centrifuge briefly. Use a magnetic rack to remove the supernatant. Repeat once, and wash twice with rinsing solution 2. (7) Add 1 mL of anhydrous ethanol, mix well and centrifuge briefly, then use a magnetic rack to adsorb and discard the supernatant; (8) After opening the centrifuge tube cap, place it in a 65°C metal bath to dry the magnetic beads, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate at 65°C for 5 minutes. (9) Centrifuge briefly to concentrate the liquid to the bottom of the tube, then place it in a magnetic rack and let it stand until the solution becomes clear; (10) Transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0099] 3. Construction of small RNA library from seminal plasma samples: (1) Template denaturation: The RNA sample and the diluted 3' adapter were mixed and denatured at 70°C for 2 min, and then placed on ice; (2) 3' connector connection: Add 3' connector buffer and 3' connector enzyme mix to the product from the previous step, mix well, and incubate at 25°C for 1 hour; (3) Blocking extra 3' linkers with reverse transcription primers: Add diluted reverse transcription primers to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 75℃, 5 min; 37℃, 15 min; 25℃, 15 min; (4) 5' connector denaturation: The diluted 5' connector was denatured at 70°C for 2 min and then placed on ice; (5) 5' connector connection: Add 5' connector, 5' connector buffer and 5' connector Enzymemix to the product of step (3), mix well and incubate at 25°C for 1 h; (6) Reverse transcription: Add reverse transcription buffer and reverse transcription enzyme mix to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 50℃ for 1 h; 80℃ for 5 min; (7) PCR library amplification: Add PCR amplification mix, universal primers and index primers to the product from the previous step, mix well and place on a PCR instrument for reaction. The reaction program is 94℃ denaturation for 3 min; 94℃, 15 s, 65℃, 15 s, 72℃, 15 s, for a total of 22-25 cycles; 72℃, 1 min.

[0100] PCR products were purified using a silica gel membrane purification column to obtain a pure library. The FragmentAnalyzer quality control results are as follows: Figure 9 As shown.

[0101] Example 5 1. The reagent preparation ratio in this example is: (1) Ligation binding solution: 75 mM Tris-HCl (pH 7.5), 1.5% (wt / vol) SDS, 1.5 mM EDTA, 150 mM NaCl, 37.5 mM sodium citrate, 1.5 M guanidine isothiocyanate; (2) Rinse solution 1: lysis binding solution: anhydrous ethanol ratio of 2:3 (vol / vol); (3) Rinse solution 2: ddH2O: anhydrous ethanol is 1:4 (vol / vol).

[0102] 2. Using the above-mentioned lysis binding buffer, small RNA was extracted from serum samples of 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, and 700 μL, respectively, according to sample volume gradients. The steps included: (1) After mixing the serum sample, centrifuge it, add 2 times the sample volume of lysis binding buffer, and vortex to mix. (2) Add 0.1 times the sample volume of proteinase K and vortex to mix; (3) Add 0.15 times the sample volume of purified magnetic beads, vortex to mix, and incubate at 60°C for 10 min; (4) Add 5.9 times the volume of anhydrous ethanol, vortex mix, and discard the supernatant after the magnetic beads are adsorbed by the magnetic rack. (5) Add 1 mL of rinsing solution 1, vortex to mix, transfer all liquid to a new EP tube, and use a magnetic rack to adsorb and discard the supernatant; (6) Add 1 mL of rinsing solution 2, mix well and centrifuge briefly. Use a magnetic rack to remove the supernatant. Repeat once, and wash twice with rinsing solution 2. (7) Add 1 mL of anhydrous ethanol, mix well and centrifuge briefly, then use a magnetic rack to adsorb and discard the supernatant; (8) After opening the centrifuge tube cap, place it in a 65°C metal bath to dry the magnetic beads, add 15 μL of elution buffer, mix at 1500 rpm for 15 seconds, and then incubate at 65°C for 5 minutes. (9) Centrifuge briefly to concentrate the liquid to the bottom of the tube, then place it in a magnetic rack and let it stand until the solution becomes clear; (10) Transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent library construction experiments or placed in a container. Store at 80℃ for later use.

[0103] 3. Construction of small RNA library from serum samples: (1) Template denaturation: The RNA sample and the diluted 3' adapter were mixed and denatured at 70°C for 2 min, and then placed on ice; (2) 3' connector connection: Add 3' connector buffer and 3' connector enzyme mix to the product from the previous step, mix well, and incubate at 25°C for 1 hour; (3) Blocking extra 3' linkers with reverse transcription primers: Add diluted reverse transcription primers to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 75℃, 5 min; 37℃, 15 min; 25℃, 15 min; (4) 5' connector denaturation: The diluted 5' connector was denatured at 70°C for 2 min and then placed on ice; (5) 5' connector connection: Add 5' connector, 5' connector buffer and 5' connector Enzymemix to the product of step (3), mix well and incubate at 25°C for 1 h; (6) Reverse transcription: Add reverse transcription buffer and reverse transcription enzyme mix to the product from the previous step, mix well, and then place on a PCR instrument for reaction. The reaction program is 50℃ for 1 h; 80℃ for 5 min; (7) PCR library amplification: Add PCR amplification mix, universal primers and index primers to the product from the previous step, mix well and place on a PCR instrument for reaction. The reaction program is 94℃ denaturation for 3 min; 94℃, 15 s, 65℃, 15 s, 72℃, 15 s, for a total of 22-25 cycles; 72℃, 1 min.

[0104] PCR products were purified using a silica gel membrane purification column to obtain a pure library. The FragmentAnalyzer quality control results are as follows: Figure 10 .

[0105] Example 6 Based on the library sequencing results, hsa-miR-16-5p was selected as the detection marker, and the library consistency was verified by real-time quantitative fluorescence PCR.

[0106] The specific sequences are shown in Table 1.

[0107] Table 1

[0108] A commercially available one-step qRT-PCR kit was used, and the reaction system is shown in Table 2.

[0109] Table 2

[0110] The reaction procedure is shown in Table 3.

[0111] Table 3

[0112] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An extraction reagent for extracting small RNAs from circulating body fluids, characterized in that, The small RNA extraction reagent includes a lysis binding buffer, which comprises Tris-HCl at a final concentration of 60-80 mmol / L, SDS at a mass / volume percentage of 1-3%, EDTA at a concentration of 1-3 mmol / L, NaCl at a concentration of 130-150 mmol / L, sodium citrate at a concentration of 30-40 mmol / L, and guanidine isothiocyanate at a concentration of 1-3 mol / L.

2. The small RNA extraction reagent according to claim 1, characterized in that, The small RNA extraction reagent further includes rinsing solution 1 and rinsing solution 2; wherein, rinsing solution 1 is prepared by mixing the lysis binding solution and anhydrous ethanol, and the content of anhydrous ethanol in rinsing solution 1 is 60%-75% by volume percentage; rinsing solution 2 is prepared by mixing ddH2O and anhydrous ethanol at a volume ratio of 1:(3-5), preferably 1:

4.

3. The small RNA extraction reagent according to claim 1 or 2, characterized in that, The circulating body fluids include blood, serum, plasma, urine, exosomes, or semen.

4. An extraction kit for extracting small RNA from circulating body fluids, characterized in that: The small RNA extraction kit includes the small RNA extraction reagents shown in any one of claims 1-3.

5. The use of the small RNA extraction reagent according to any one of claims 1-3 or the small RNA extraction kit according to claim 4 in the extraction of small RNA from circulating body fluids.

6. A method for extracting small RNA from circulating body fluids, characterized in that, Includes the following steps: 1) The circulating body fluid sample is vortexed and mixed with the lysis binding solution according to any one of claims 1-3; 2) Add proteinase K solution and vortex to mix; 3) Add the purified magnetic beads and vortex mix well; 4) Constant temperature incubation; 5) After incubation, add anhydrous ethanol, vortex to mix, let stand until clear, and then discard the supernatant. 6) Add the rinse solution 1 and mix well. Centrifuge briefly, let stand until clear, and then discard the supernatant. 7) Repeat the addition of the rinse solution 1, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 8) Add the rinse solution 2 and mix well. Centrifuge briefly, let stand until clear, and then discard the supernatant. 9) Repeat the addition of the rinsing solution 2, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 10) Add anhydrous ethanol, mix well, centrifuge briefly, let stand until clear, and then discard the supernatant; 11) Centrifuge briefly and remove residual liquid; 12) After drying in a metal bath, add ddH2O and mix well, then incubate in a metal bath. 13) After instantaneous centrifugation, allow to stand until clear; 14) The supernatant is the small RNA extracted from the circulating body fluid.

7. The method for extracting small RNA from circulating body fluids according to claim 6, characterized in that, The ratio of the circulating body fluid sample, lysis binding buffer, proteinase K solution, purified magnetic beads, and anhydrous ethanol was 1:(1.8-2.2):(0.08-0.12):(0.1-0.2):(4.8-5.9) by volume. The mixing time for steps 1) and 2) is 5-10 seconds, preferably 5 seconds; The mixing time for steps 3), 5), 6), 7), 8), 9), 10), and 12) is 10-15 seconds, preferably 15 seconds; In step 4), the incubation is carried out by water bath incubation at 60-65℃ or constant temperature forced air drying incubation, preferably at 60℃; the incubation time is 10-15 minutes, preferably 10 minutes; In steps 6) and 7), the amount of rinsing solution 1 added is 0.8-1.2 ml / time, preferably 1 ml; In steps 8) and 9), the amount of rinsing solution 2 added is 0.8-1.2 ml / time, preferably 1 ml; In steps 5)-10), the settling time is 2-5 minutes; In step 12), the temperature for drying and incubating the metal bath is 60℃-65℃, preferably 65℃, the amount of eluent added is 10-30μL, preferably 15μL, and the mixture is stirred at 1500rpm for 15-30 seconds, preferably 15 seconds. The incubation time in the metal bath is 5-10 minutes, preferably 5 minutes. In step 13), the settling time is 2-5 minutes.

8. A method for constructing a library of small RNAs in circulating body fluids, characterized in that, Includes the following steps: a) Small RNAs in circulating body fluids are extracted using the extraction method of claim 6 or 7; b) Linking 3' adapters to small RNAs in circulating body fluids; c) Seal off any excess 3' connector; d) Ligate 5' adapters to small RNAs in circulating body fluids; e) Reverse transcription of small RNAs in circulating body fluids into cDNA; f) After PCR amplification and purification of the PCR products, sequencing libraries of small RNAs in circulating body fluids were obtained by enrichment.

9. The database construction method according to claim 8, characterized in that, The dilution ratio of the 3' adapter, reverse transcription primer and 5' adapter is calculated by volume ratio (adapter / primer):ddH2O=1:(15-20), preferably 1:

19.

10. The database construction method according to claim 9, characterized in that, In step f), the amount of universal primers and index primers added is 0.5-1 μL, preferably 0.5 μL, and the PCR amplification program is 20-25 cycles, preferably 25 cycles.

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