A reagent for extracting small RNAs, its method and application

Abstract

The present invention relates to a reagent for extracting small RNAs, a method thereof and an application. The reagent for extracting small RNAs includes a lysis solution, and the lysis solution includes 1-10 M guanidine isothiocyanate, 0.1-1 M ammonium thiocyanate, and 50-300 mM Tris; the reagent further includes a washing solution, and the washing solution includes 5-50 mM Tris, 5-50 mM NaCl, and 65%-75% absolute ethanol. Using the reagent provided by the present invention to extract small RNAs from biological fluids has a high yield, high purity, is easy to operate, and has a low cost.

Description

Technical Field

[0001] The present invention relates to nucleic acid detection technology, and particularly to a reagent for extracting small RNAs, a method thereof and an application thereof. Background Art

[0002] Small RNAs are a large class of regulatory molecules that exist in almost all organisms. Small RNAs include: miRNAs, siRNAs, snoRNAs, piRNAs, etc. Small RNAs are involved in post-transcriptional gene expression regulation. A large amount of data shows that small RNAs are involved in the abnormal expression of proteins in various disease patients through gene regulation. As the influence of small RNAs on biological pathways becomes greater and changes the biological states of many genes, the extraction and separation of small RNAs have attracted more and more attention from researchers in recent years.

[0003] The extraction method and process standardization of small RNAs are key factors in ensuring the quality of small RNAs. High-purity and high-yield small RNA extraction methods or kits help to ensure high-quality and accurate downstream research applications. In recent years, many scientific researchers have conducted research on small RNA extraction methods. There are various small RNA extraction methods, and the currently commonly used ones are mainly precipitation methods and filtration column methods. The precipitation method mainly uses the TRIZOL extraction method to directly extract small RNAs from cells or tissues. TRIZOL contains substances such as phenol and guanidine isothiocyanate, which can quickly break cells and inhibit nucleases released by cells. However, this method requires the use of phenol, which is harmful to the human body, and the extraction efficiency is low, the sample volume requirement is large, and the residue of organic reagents will inhibit subsequent PCR reactions; the filtration column method mainly uses centrifugation to extract small RNAs, and uses the principle of high-salt adsorption and low-salt elution to extract small RNAs. However, small RNA fragments are widely distributed, about 15 - 200 bp, and are not easily adsorbed and precipitated, and this method usually causes loss of small RNAs and destroys the integrity of RNAs.

[0004] At present, there is no relatively fast and efficient method for small RNA extraction. Therefore, there is an urgent need in the art to develop a method for efficiently, accurately and highly purely extracting small RNAs from biological fluids. Summary of the Invention

[0005] In view of the technical defects existing in the prior art, the present invention provides a small RNA extraction reagent and a usage method with high extraction rate, high purity, wide sample adaptability (such as blood, serum, saliva, urine, plasma, exosomes, etc.) and strong anti-interference ability.

[0006] In the first aspect of the present invention, a reagent for extracting small RNAs is provided, and the reagent includes a lysis solution, and the lysis solution includes 1 - 10 M guanidine isothiocyanate, 0.1 - 1 M ammonium thiocyanate, and 50 - 300 mM Tris;

[0007] The reagent further includes a washing solution, which includes 5 - 50 mM Tris, 5 - 50 mM NaCl, and 65% - 75% absolute ethanol.

[0008] Preferably, the concentration of guanidine isothiocyanate is 4 - 6 M, the concentration of ammonium thiocyanate is 0.1 - 0.5 M, and the concentration of Tris is 50 - 200 mM.

[0009] Preferably, the pH of the lysis solution is 6.5 - 7.5.

[0010] Preferably, the reagent includes an adsorption solution, which is composed of SiC adsorption beads and the lysis solution, and the concentration of SiC adsorption beads is 0.2 - 1 g / mL.

[0011] Preferably, the particle size of the SiC adsorption beads is 4 - 10 μm, and the purity is greater than 95%.

[0012] Preferably, the concentration of NaCl in the washing solution is 10 - 50 mM, and the volume fraction of absolute ethanol is 70% - 75%.

[0013] The second aspect of the present invention provides a kit containing the reagent described in any one of the above.

[0014] The third aspect of the present invention provides a method for extracting small RNA using the reagent described in any one of the above, and the method includes the following steps:

[0015] (1) Mix the sample to be extracted, the lysis solution, and the adsorption solution to obtain a mixed solution A, and incubate.

[0016] (2) After the incubation is completed, mix the mixed solution A with absolute ethanol, centrifuge, discard the supernatant, and obtain a mixed solution B.

[0017] (3) Transfer the mixed solution B to a silica membrane column and centrifuge.

[0018] (4) Add the washing solution to the silica membrane column, centrifuge, add the elution solution to the silica membrane column, and elute the small RNA to obtain the small RNA.

[0019] Preferably, in the step (1), the volume ratio of the sample to the lysis solution is 1:(1.8 - 2.2).

[0020] Preferably, the volume ratio of the sample to the adsorption solution is 1:(0.05 - 0.2).

[0021] Preferably, the temperature of the incubation is 50 - 65°C, and the time of the incubation is 5 - 10 min.

[0022] Preferably, the sample is selected from blood, serum, saliva, urine, plasma, cerebrospinal fluid, follicular fluid, allantoic fluid, interstitial fluid, endolymph, pericardial fluid, ventricular fluid, serous fluid, synovial fluid, tissue fluid, milk, semen, ocular fluid, amniotic fluid, placental fluid, ascites, pleural effusion, sputum, bronchial aspirate, and exosomes.

[0023] Preferably, in step (2), the volume of the absolute ethanol is 2 - 4 times the volume of the sample.

[0024] Preferably, between step (2) and step (3), the following steps are further included: mixing the mixture B with the lysis solution, and then mixing with absolute ethanol to obtain a mixture C, and subjecting the mixture C to step (3).

[0025] Preferably, in step (4), the eluent is DEPC water or nuclease - free water.

[0026] Preferably, in step (4), the volume of the eluent is 30 - 100 μL.

[0027] The fourth aspect of the present invention provides the application of the reagent according to any one of the above in extracting small RNAs.

[0028] Compared with the prior art, the present invention has the following technical effects:

[0029] The reagent for extracting small RNAs provided by the present invention includes a lysis solution with specific components, which can fully and effectively lyse cells with high lysis efficiency. With the cooperation of other reagents, small RNAs in the sample can be obtained quickly and efficiently, and the obtained small RNAs have high purity and high yield, which is convenient for subsequent experiments (such as RT - qPCR, second - generation sequencing library construction, etc.); moreover, the extraction method provided by the present invention has the advantages of simple operation, low cost, green and safe, and has broad application prospects and great market promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the quality detection result of extracting small RNAs by the Qiagen kit in Example 1 of the present invention;

[0031] Figure 2 It is the quality detection result of extracting small RNAs by the method provided by the present invention in Example 1 of the present invention;

[0032] Figure 3 It is the quality detection result of extracting small RNAs by the Qiagen kit in Example 2 of the present invention;

[0033] Figure 4 It is the quality detection result of extracting small RNAs by the method provided by the present invention in Example 2 of the present invention;

[0034] Figure 5 The peak graph of constructing a second-generation sequencing library after extracting small RNAs using the Qiagen kit in Example 2 of the present invention;

[0035] Figure 6 The peak graph of constructing a second-generation sequencing library after extracting small RNAs using the method provided by the present invention in Example 2 of the present invention;

[0036] Figure 7 The quality detection result of extracting small RNAs using the Qiagen kit in Example 3 of the present invention;

[0037] Figure 8 The quality detection result of extracting small RNAs using the method provided by the present invention in Example 3 of the present invention;

[0038] Figure 9 The column graph of CT comparison after RT-qPCR of extracting small RNAs using the Qiagen kit and the method provided by the present invention in Example 3;

[0039] Figure 10 The quality detection result of extracting small RNAs using the Qiagen kit in Example 4 of the present invention;

[0040] Figure 11 The quality detection result of extracting small RNAs using the method provided by the present invention in Example 4 of the present invention;

[0041] Figure 12 The quality detection result of extracting small RNAs using the Novoprotein kit in Example 4 of the present invention;

[0042] Figure 13 The quality detection result of extracting small RNAs using the method provided by the present invention in Example 4 of the present invention. Detailed implementation manners

[0043] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0044] High-quality RNA is the first and crucial step in molecular biology research, directly affecting the success or failure of experiments. With the increasingly wide clinical application of small RNAs in biological fluids (such as plasma or serum), the extraction methods for high-quality and high-concentration small RNAs in biological fluids are monopolized by foreign countries, which has affected the development of small RNA research in biological fluids in our country. Based on this, the inventor of the present invention has proposed a method for extracting small RNAs from biological fluids. After forming a kit, this method can be used for the extraction of small RNAs in biological fluids such as cells, serum, plasma, exosomes, urine, etc. The products extracted by this kit can be directly applied to the detection of small RNAs, such as RT-qPCR, next-generation sequencing library construction, etc. The reagents and extraction methods provided by the present invention have the advantages of high yield, high purity, wide sample applicability, strong anti-interference ability, and a wide range of nucleic acid fragment sizes for extraction.

[0045] In the first aspect of the present invention, a reagent for extracting small RNAs is provided. The reagent includes a lysis solution, and the lysis solution includes 1-10 M (such as 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, etc.) guanidine isothiocyanate, 0.1-1 M (such as 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, etc.) ammonium thiocyanate, and 50-300 mM (such as 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, etc.) Tris;

[0046] The reagent further includes a washing solution, which includes 5 - 50 mM (such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, etc.) Tris, 5 - 50 mM (such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, etc.) NaCl, and 65% - 75% (such as 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, etc.) absolute ethanol.

[0047] In certain embodiments, the lysis solution consists of 1 - 10 M guanidine isothiocyanate, 0.1 - 1 M ammonium thiocyanate, and 50 - 300 mM Tris.

[0048] In certain embodiments, the concentration of guanidine isothiocyanate is 4 - 6 M, the concentration of ammonium thiocyanate is 0.1 - 0.5 M, and the concentration of Tris is 50 - 200 mM.

[0049] In certain embodiments, the pH of the lysis solution is 6.5 - 7.5.

[0050] In certain embodiments, the pH of the lysis solution is 6.9 - 7.5.

[0051] In some embodiments, the reagent includes an adsorption solution, which is composed of SiC adsorption beads and the lysis solution, and the concentration of the SiC adsorption beads is 0.2 - 1 g / mL. In this application, the SiC adsorption beads are fixed - volume with the lysis solution to form the adsorption solution, which can more fully bind and extract the nucleic acids released in the supernatant, so as to further improve the nucleic acid extraction yield and purity of the sample. At the same time, the cost is saved, and there is no need to select other reagents as the adsorption solution.

[0052] In some embodiments, the particle size of the SiC adsorption beads is 4 - 10 μm, and the purity is greater than 95%.

[0053] In some embodiments, the washing solution is composed of 5 - 50 mM Tris, 5 - 50 mM NaCl, and 65% - 75% absolute ethanol.

[0054] In this application, 65% - 75% absolute ethanol refers to absolute ethanol with a volume fraction of 65% - 75%.

[0055] In some embodiments, the NaCl concentration in the washing solution is 10 - 50 mM, and the concentration of the absolute ethanol is 70% - 75%.

[0056] In the second aspect, the present invention provides a kit containing the reagent described in any one of the above.

[0057] In the third aspect, the present invention provides a method for extracting small RNAs using the reagent described in any one of the above. The method includes the following steps:

[0058] (1) Mix the sample to be extracted, the lysis solution, and the adsorption solution to obtain a mixed solution A, and incubate.

[0059] (2) After incubation, mix the mixed solution A with absolute ethanol, centrifuge, discard the supernatant, and obtain a mixed solution B.

[0060] (3) Transfer the mixed solution B to a silica membrane column and centrifuge.

[0061] (4) Add the washing solution to the silica membrane column, centrifuge, add the elution solution to the silica membrane column, and elute the small RNAs to obtain the small RNAs.

[0062] In some embodiments, in step (1), the volume ratio of the sample to the lysis solution is 1:(1.8 - 2.2), such as 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2.

[0063] In some embodiments, the volume ratio of the sample to the adsorption solution is 1:(0.05 - 0.2), such as 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19, 1:0.2, etc.

[0064] In some embodiments, the temperature of the incubation is 50 - 65 °C (such as 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, etc.), and the time of the incubation is 5 - 10 min (such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.).

[0065] In some embodiments, the sample is selected from blood, serum, saliva, urine, plasma, cerebrospinal fluid, follicular fluid, allantoic fluid, interstitial fluid, endolymph, pericardial fluid, ventricular fluid, serous fluid, synovial fluid, tissue fluid, milk, semen, ocular fluid, amniotic fluid, placental fluid, ascites, pleural effusion, sputum, bronchial aspirate, and exosomes.

[0066] In some embodiments, the sample is selected from blood, serum, saliva, urine, plasma, and exosomes.

[0067] In some embodiments, in step (2), the volume of the absolute ethanol is 2 - 4 times the volume of the sample, such as 2 times, 2.5 times, 3 times, 3.5 times, 4 times.

[0068] In some embodiments, between step (2) and step (3), the following steps are further included: mixing the mixture B with the lysis solution, and then mixing with the absolute ethanol to obtain a mixture C, and subjecting the mixture C to step (3).

[0069] In some embodiments, step (3) can be repeated multiple times (such as 1 time, 2 times, 3 times, 4 times, 5 times). In a preferred embodiment, step (3) is repeated 2 times.

[0070] In some embodiments, in step (4), the eluent is DEPC water or nuclease-free water.

[0071] In some embodiments, in step (4), the volume of the eluent is 30 - 100 μL, such as 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, etc.

[0072] In certain embodiments, the lysis solution comprises 1-10 M (such as 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, etc.) guanidine isothiocyanate, 0.1-1 M (such as 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, etc.) ammonium thiocyanate, and 50-300 mM (such as 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, etc.) Tris.

[0073] In certain embodiments, the concentration of guanidine isothiocyanate is 4-6 M guanidine isothiocyanate, 0.1-0.5 M ammonium thiocyanate, and 50-200 mM Tris.

[0074] In certain embodiments, the pH of the lysis solution is 6.5-7.5.

[0075] In certain embodiments, the adsorption solution consists of SiC adsorption beads and the lysis solution, and the concentration of the SiC adsorption beads is 0.2-1 g / mL.

[0076] In certain embodiments, the particle size of the SiC adsorption beads is 4-10 μm, and the purity is greater than 95%.

[0077] In some embodiments, the washing solution comprises 5 - 50 mM (such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, etc.) Tris, 5 - 50 mM (such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, etc.) NaCl and 65% - 75% (such as 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, etc.) absolute ethanol.

[0078] In some embodiments, the concentration of NaCl in the washing solution is 10 - 50 mM, and the volume fraction of the absolute ethanol is 70% - 75%.

[0079] The fourth aspect of the present invention provides the application of the reagent described in any one of the above in extracting small RNAs.

[0080] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Example 1

[0081] Reagent preparation for this example:

[0082] (1) Lysis buffer: 5 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0;

[0083] (2) Adsorption solution: Dilute the adsorption beads (SiC) with the lysis buffer to a mass concentration of 1 g / mL for SiC. Among them, the particle size of the adsorption beads is 5 μm and the purity is 95.5%;

[0084] (3) Wash buffer: 15 mM Tris, 15 mM NaCl, 73% absolute ethanol.

[0085] Using the above reagents to extract small RNAs from whole blood samples, the steps include:

[0086] (1) Mix the whole blood sample and then centrifuge. Based on the sample volume, add 1.9 times the volume of the lysis buffer and 0.1 times the volume of the adsorption solution to obtain mixture 1, and incubate at 55 °C for 5 min;

[0087] (2) Add 3 times the sample volume of absolute ethanol to the mixture 1, centrifuge briefly, discard the supernatant to obtain mixture 2;

[0088] (3) Add 350 μL of the lysis buffer to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0089] (4) Transfer the mixture 3 to a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12000 g for 1 min;

[0090] (5) Add 500 μL of the wash buffer to the silica membrane column, centrifuge at 12000 g for 1 min, and repeat 2 times;

[0091] (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min;

[0092] (7) Collect the eluate with a nuclease-free centrifuge tube and centrifuge at 13000 g for 2 min;

[0093] (8) Detect small RNAs.

[0094] At the same time, in this example, the miRNA extraction kit from Qiagen (Qiagen, miRNeasy Serum / Plasma Advanced Kit, catalog number: 217204) was used to extract small RNAs for comparison.

[0095] The extracted products were subjected to quality testing using a small RNA kit of Agilent brand. The quality testing results of small RNA extracted using the Qiagen kit are as Figure 1 shown, and the quality testing results of small RNA extracted by the method provided by the present invention are as Figure 2 shown. Among them, compared with Figure 1 , taking the 4bp peak as a reference, it can be seen that the nucleic acid concentration of the product with 20 - 40bp obtained by the method provided by the present invention is higher and the proportion is larger. Therefore, the product obtained by the method provided by the present invention has a higher concentration and has an obvious peak after 150bp, indicating better abundance of small RNA. Example 2

[0096] Reagent preparation in this example:

[0097] (1) Lysis solution (lysis buffer): 5.5 M guanidine isothiocyanate, 0.3 M ammonium thiocyanate, 50 mM Tris, pH = 6.9;

[0098] (2) Adsorption solution: Dilute the adsorption beads (SiC) with the lysis solution to a mass concentration of SiC of 0.75 g / mL. Among them, the particle size of the adsorption beads is 5 μm and the purity is 95%;

[0099] (3) Washing solution: 25 mM Tris, 25 mM NaCl, 71% absolute ethanol.

[0100] Using the above reagents to extract small RNA from plasma samples, the steps include:

[0101] (1) Mix the plasma sample and centrifuge it. Based on the sample volume, add 1.8 times the volume of the lysis solution and 0.2 times the volume of the adsorption solution to obtain a mixed solution 1, and incubate it at 56 °C for 10 min;

[0102] (2) Add 3 times the sample volume of absolute ethanol to the mixed solution 1, centrifuge briefly, and discard the supernatant to obtain a mixture 2;

[0103] (3) Add 350 μL of the lysis solution to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain a mixed solution 3;

[0104] (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12000 g for 1 min;

[0105] (5) Add 500 μL of the washing solution to the silica membrane column, centrifuge at 12000 g for 1 min, and repeat 2 times;

[0106] (6) Add 50 μL of DEPC water into the silica membrane column and let it stand for 2 min;

[0107] (7) Collect the eluate using a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min;

[0108] (8) Detect small RNAs.

[0109] Meanwhile, in this example, the small RNAs were extracted using the QIAGEN miRNeasy Serum / Plasma Advanced Kit (Qiagen, miRNeasy Serum / Plasma Advanced Kit, catalog number: 217204) for comparison.

[0110] The extracted products were subjected to quality detection using an Agilent small RNA kit, and the downstream next-generation sequencing library was constructed using the miRNA Library Kit (Qiagen, QIAseq miRNA Library Kit). The library construction and quality inspection methods can be found in the instruction manual of the QIAGEN miRNA Library Kit (Qiagen, QIAseq miRNA Library Kit). The quality detection results of the small RNAs extracted using the Qiagen kit are as Figure 3 shown, and the quality detection results of the small RNAs extracted using the method provided by the present invention are as Figure 4 shown. By comparing with Figure 3 and taking the 4-bp peak as a reference, it can be seen that the nucleic acid concentration of 20 - 40 bp obtained by the method provided by the present invention is higher and the proportion is larger; the peak map of the next-generation sequencing library constructed after extracting small RNAs using the Qiagen kit is shown in Figure 5 , and the peak map of the next-generation sequencing library constructed after extracting small RNAs using the method provided by the present invention is shown in Figure 6 From Figure 6 it can be seen that the quality of the products obtained by the method provided by the present invention is higher, and the small RNAs obtained using the method provided by the present invention can be directly applied to the construction of next-generation sequencing libraries. Example 3

[0111] Reagent preparation in this example:

[0112] (1) Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 55 mM Tris, pH = 7.1;

[0113] (2) Adsorption solution: The adsorption beads (SiC) were fixed to a mass concentration of 1 g / mL with the lysis solution. Among them, the particle size of the adsorption beads is 7.5 μm and the purity is 95%;

[0114] (3) Wash solution: 25 mM Tris, 25 mM NaCl, 75% absolute ethanol.

[0115] Using the above reagents to extract small RNAs from serum samples, the steps include:

[0116] (1)Add 5 μL of 50 nM cel-39 (purchased from Qiagen) to each milliliter of serum, mix well and centrifuge briefly. Then, based on the sample volume, add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution to obtain mixture 1, and incubate at 56 °C for 10 min;

[0117] (2)Add 3 times the sample volume of absolute ethanol to the mixture 1, centrifuge briefly, and discard the supernatant to obtain mixture 2;

[0118] (3)Add 350 μL of lysis solution to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0119] (4)Transfer the mixture 3 to a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12,000 g for 1 min;

[0120] (5)Add 500 μL of wash solution to the silica membrane column, centrifuge at 12,000 g for 1 min, and repeat 2 times;

[0121] (6)Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min;

[0122] (7)Collect the eluate with a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min;

[0123] (8)Detect small RNAs.

[0124] Meanwhile, in this example, the Qiagen miRNeasy Serum / Plasma Advanced Kit was used to extract small RNAs for comparison.

[0125] The extracted products were subjected to quality detection using an Agilent small RNA kit, and several small RNAs (Cel-39, miR-142-3P, miR-26b) were selected for concentration comparison using the qPCR method with Thermo Fisher reagents. The steps of miRNA reverse transcription were carried out according to the instructions of the kit (TaqMan MicroRNA Reverse Transcription Kit, catalog number: 4366596), the pre-amplification method was carried out according to the Thermo Fisher pre-amplification kit (TaqMan PreAmp Master Mix, catalog number: 4384266), and real-time quantitative PCR was carried out using the Thermo Fisher qPCR kit (TaqMan Fast Advanced Master Mix, catalog number: 4444557). The results are shown in Figures 7 to 9 .

[0126] The quality test results of small RNAs extracted by the Qiagen kit are as follows Figure 7 shown, and the quality test results of small RNAs extracted by the method provided by the present invention are as follows Figure 8 shown. Among them, compared with Figure 7 , taking the 4bp peak as a reference, it can be seen that the product obtained by the method provided by the present invention has a higher nucleic acid concentration of 20-40bp and a larger proportion. Therefore, the product obtained by the method provided by the present invention has a higher concentration. In addition, there is an obvious peak after 150bp, indicating better abundance of small RNAs.

[0127] Figure 9 are the CT bar graphs of RT-qPCR after extracting small RNAs by the Qiagen kit and the method provided by the present invention. Among them, Q1 and Q2 are the CT bar graphs of RT-qPCR after extracting RNA by the Qiagen kit, and ZY1 and ZY2 are the CT bar graphs of RT-qPCR after extracting RNA by the kit of the present invention. From the CT values, the CT of the kit of the present invention is smaller and better. Example 4

[0128] Reagent preparation in this example:

[0129] (1) Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 55 mM Tris, pH = 7.0;

[0130] (2) Adsorption solution: Dilute the adsorption beads (SiC) with the lysis solution to a mass concentration of SiC of 1 g / mL. Among them, the particle size of the adsorption beads is 5 μm and the purity is 95%;

[0131] (3) Wash solution: 25 mM Tris, 25 mM NaCl, 73% absolute ethanol.

[0132] Using the above reagents to extract small RNAs from exosome samples, the steps include:

[0133] (1) Extract plasma exosomes using the MARCKS-ED magnetic bead exosome enrichment and purification kit of Ehang (Suzhou) Biotechnology Co., Ltd. (refer to CN115558629A);

[0134] (2) Based on the sample volume, add 1.9 times the volume of the lysis buffer and 0.1 times the volume of the adsorption solution to the sample to obtain mixture 1, and incubate at 56 °C for 10 min;

[0135] (3) Add 3 times the sample volume of absolute ethanol to the mixture 1, briefly centrifuge, discard the supernatant, and obtain mixture 2;

[0136] (4) Add 350 μL of the lysis solution to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0137] (5) Transfer the mixture 3 into a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12,000 g for 1 min;

[0138] (6) Add 500 μL of wash buffer into the silica membrane column, centrifuge at 12,000 g for 1 min, and repeat 2 times;

[0139] (7) Add 100 μL of DEPC water into the silica membrane column, and let it stand for 2 min;

[0140] (8) Collect the eluate with a nuclease-free centrifuge tube, and centrifuge at 13,000 g for 2 min;

[0141] (9) Detect small RNAs.

[0142] Meanwhile, in this example, the small RNAs were extracted using the QIAGEN miRNeasy Serum / Plasma Advanced Kit (Qiagen) for comparison.

[0143] The quality of the extracted products was detected using an Agilent brand small RNA kit. The quality detection results of the small RNAs extracted by the Qiagen kit are as Figure 10 shown, and the quality detection results of the small RNAs extracted by the method provided by the present invention are as Figure 11 shown. From Figure 10 comparison, it can be seen that the product concentration obtained by the method provided by the present invention is higher, and there is an obvious peak after 150 bp, indicating better abundance of small RNAs ( Figure 11 ). Example 5

[0144] Reagent preparation in this example:

[0145] (1) Lysis buffer: 5 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0;

[0146] (2) Adsorption solution: Dilute the adsorption beads (SiC) with the lysis buffer to a mass concentration of 1 g / mL of SiC. Among them, the particle size of the adsorption beads is 7.5 μm, and the purity is 95%;

[0147] (3) Wash buffer: 50 mM Tris, 50 mM NaCl, 75% absolute ethanol.

[0148] The steps for extracting small RNAs from urine samples using the above reagents include:

[0149] (1) Mix the urine sample evenly and centrifuge it. Calculate based on the sample volume and add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution to obtain mixture 1, and incubate it at 55 °C for 10 min;

[0150] (2) Add 3 times the sample volume of absolute ethanol to the mixture 1, centrifuge briefly, discard the supernatant to obtain mixture 2;

[0151] (3) Add 350 μL of lysis solution to the mixture 2, then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0152] (4) Transfer the mixture 3 to a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12000 g for 1 min;

[0153] (5) Add 500 μL of washing solution to the silica membrane column, centrifuge at 12000 g for 1 min, and repeat 2 times;

[0154] (6) Add 100 μL of DEPC water into the silica membrane column and let it stand for 2 min;

[0155] (7) Collect the eluate with a nuclease-free centrifuge tube and centrifuge at 13000 g for 2 min;

[0156] (8) Detect small RNAs.

[0157] Meanwhile, in this example, the Trizol method was used to extract RNA, and the reagents used were purchased from Novoprotein (product number: R401-01) as a control.

[0158] The extracted products were subjected to quality detection using a small RNA kit of the Agilent brand. The quality detection results of the small RNAs extracted by the Novoprotein kit are as Figure 12 shown, and the quality detection results of the small RNAs extracted by the method provided by the present invention are as Figure 13 shown. Compared with Figure 12 it can be seen that the product concentration obtained by the method provided by the present invention is higher. Example 6

[0159] In this example, other lysis solution formulations were selected and used in combination with the adsorption solution and washing solution of the present invention to extract small RNAs. The specific lysis solution formulations are shown in Table 1:

[0160] Table 1

[0161]

[0162] Adsorption solution: Dilute the adsorption beads (SiC) with the lysis solution to a mass concentration of 1 g / mL for SiC. Among them, the particle size of the adsorption beads is 7.5 μm and the purity is 95%;

[0163] Washing solution: 50 mM Tris, 50 mM NaCl, 75% absolute ethanol.

[0164] The small RNAs in plasma and serum samples were extracted using the above reagents respectively, and the operation steps were as follows:

[0165] (1) Add 5 μL of 50 nM cel-39 (purchased from Qiagen) to each milliliter of serum or plasma. After mixing, perform a simple centrifugation. Based on the sample volume, add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution to obtain mixture 1, and incubate at 55 °C for 10 min;

[0166] (2) Add 3 times the sample volume of absolute ethanol to the mixture 1, perform a short centrifugation, and discard the supernatant to obtain mixture 2;

[0167] (3) Add 350 μL of lysis solution to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0168] (4) Transfer the mixture 3 to a silica membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12,000 g for 1 min;

[0169] (5) Add 500 μL of washing solution to the silica membrane column, centrifuge at 12,000 g for 1 min, and repeat 2 times;

[0170] (6) Add 100 μL of DEPC water into the silica membrane column and let it stand for 2 min;

[0171] (7) Collect the eluate with a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min;

[0172] (8) Detect the small RNA.

[0173] After the extracted product was detected by 2100, the concentration of small RNA was calculated. Several small RNAs (U6, Cel-39, miR-26b) were selected from the extracted product, and the concentration comparison was performed by the qPCR method using the reagents of Thermo Fisher. The steps were as described in Example 3. The detection results of extracting small RNAs from different samples using different lysis solutions are shown in Table 2:

[0174] Table 2

[0175]

[0176] As shown in the results of Table 2, the concentration of small RNA extracted using the reagents provided by the present invention (Group 1 and 2) was the highest, and the CT value after superposition was smaller, indicating that the reagents of the present invention had the best effect. Example 7

[0177] In this embodiment, other wash buffer formulations are selected and used in combination with the lysis solution and adsorption solution of the present invention to extract small RNAs. The specific wash buffer formulation is shown in Table 3:

[0178] Table 3

[0179]

[0180] Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0;

[0181] Adsorption solution: The adsorption beads (SiC) are fixed-volume with the lysis solution to a mass concentration of SiC of 1 g / mL. Among them, the particle size of the adsorption beads is 7.5 μm and the purity is 95%;

[0182] The above-mentioned reagents are used to extract small RNAs from plasma and serum samples respectively. The operation steps are as follows:

[0183] (1) Add 5 μL of 50 nM cel-39 (purchased from Qiagen) to each milliliter of serum or plasma. After mixing, perform a simple centrifugation. Based on the sample volume, add 1.9 times the volume of the lysis buffer and 0.1 times the volume of the adsorption solution to obtain mixture 1, and incubate at 55 °C for 10 min;

[0184] (2) Add 3 times the sample volume of absolute ethanol to the mixture 1, perform a short centrifugation, and discard the supernatant to obtain mixture 2;

[0185] (3) Add 350 μL of the lysis solution to the mixture 2, and then add 350 μL of absolute ethanol, mix to obtain mixture 3;

[0186] (4) Transfer the mixture 3 to a silicon membrane column (purchased from Hangzhou Xinjing), and centrifuge at 12000 g for 1 min;

[0187] (5) Add 500 μL of the wash buffer to the silicon membrane column, centrifuge at 12000 g for 1 min, and repeat 2 times;

[0188] (6) Add 100 μL of DEPC water to the silicon membrane column, and let it stand for 2 min;

[0189] (7) Collect the eluate with a nuclease-free centrifuge tube and centrifuge at 13000 g for 2 min;

[0190] (8) Detect small RNAs.

[0191] The extracted product was detected by 2100, and then the concentration of small RNA was calculated. Several small RNAs (U6, Cel-39, miR-26b) were selected from the extracted product, and the concentration was compared by qPCR method using the reagents of Thermo Fisher. The steps were as described in Example 3. The detection results of small RNAs extracted from different samples using the kits composed of different washing solutions combined with the lysis solution and adsorption solution of the present invention are shown in Table 4:

[0192] Table 4

[0193]

[0194] As can be seen from Table 4, regardless of whether the sample is plasma or serum, the concentration of small RNA extracted using the reagents provided by the present invention (Groups 7 and 8) is the highest, and the superimposed CT value is smaller, indicating that the reagents of the present invention have the best effect and a wide range of applicability to samples.

[0195] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can be made on the basis of the above embodiments. Similarly, the technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be clearly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A reagent for extracting small RNA, characterized in that: The reagent includes a lysis solution, which is composed of 5-6 M guanidine isothiocyanate, 0.1-0.3 M ammonium thiocyanate and 50-200 mM Tris; The reagent also includes a rinse solution, which consists of 5-50 mM Tris, 5-50 mM NaCl and 65%-75% anhydrous ethanol.

2. The reagent according to claim 1, characterized in that The pH of the lysate is 6.5-7.

5.

3. The reagent according to claim 1 or 2, characterized in that The reagent comprises an adsorption solution, which is composed of SiC adsorption beads and the lysis solution, and the concentration of the SiC adsorption beads is 0.2-1 g / mL.

4. The reagent according to claim 1, characterized in that The concentration of NaCl in the rinsing solution is 10-50 mM, and the concentration of anhydrous ethanol is 70-75%.

5. A method for extracting small RNA using the reagent according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) Mixing the sample to be extracted, the lysis solution and the adsorption solution to obtain a mixed solution A, and incubating; (2) After the incubation is completed, the mixed solution A is mixed with anhydrous ethanol, centrifuged, and the supernatant is discarded to obtain the mixed solution B; (3) transferring the mixed solution B to a silica membrane column and centrifuging; (4) adding a rinse solution to the silica membrane column, centrifuging, adding an elution solution to the silica membrane column, eluting the small RNA, and obtaining the small RNA.

6. The method according to claim 5, characterized in that In the step (1), the volume ratio of the sample to the lysate is 1:(1.8-2.2); and / or, the volume ratio of the sample to the adsorption liquid is 1:(0.05-0.2); And / or, the incubation temperature is 50-65°C, and the incubation time is 5-10 min; And / or, the sample is selected from blood, serum, saliva, urine, plasma, cerebrospinal fluid, follicular fluid, allantoic fluid, interstitial fluid, labyrinthine fluid, pericardial fluid, ventricular fluid, serous fluid, synovial fluid, tissue fluid, breast milk, semen, eye fluid, amniotic fluid, placental fluid, ascites, pleural effusion, sputum, bronchial aspirate and exosomes.

7. The method according to claim 5, characterized in that In step (2), the volume of the anhydrous ethanol is 2-4 times the volume of the sample; And / or, the step (2) and (3) further include the following step: mixing the mixed solution B with the lysate, and then mixing with anhydrous ethanol to obtain a mixed solution C, and subjecting the mixed solution C to step (3).

8. The method according to claim 5, characterized in that In the step (4), the elution solution is DEPC water or nuclease-free water.

9. Use of the reagent according to any one of claims 1 to 4 in extracting small RNA.

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