Reagent for extracting small RNA (Ribonucleic Acid) as well as method and application thereof

Abstract

The invention relates to a reagent for extracting small RNA (Ribonucleic Acid) as well as a method and application thereof, the reagent for extracting small RNA comprises a lysis solution, and the lysis solution comprises 1-10 M of guanidine isothiocyanate, 0.1-1 M of ammonium thiocyanate and 50-300 mM of Tris; the reagent further comprises a rinsing solution, wherein the rinsing solution comprises 5 to 50 mM of Tris, 5 to 50 mM of NaCl and 65 to 75 percent of absolute ethyl alcohol. When the reagent provided by the invention is used for extracting the small RNA in the biological fluid, the yield is high, the purity is high, the operation is easy, and the cost is low.

Description

Technical Field

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

[0002] Small RNA is a large class of regulatory molecules that exist in almost all organisms. Small RNA includes: miRNA, siRNA, snoRNA, piRNA, etc. Small RNA participates in post-transcriptional gene expression regulation. A large amount of data shows that small RNA participates in the abnormal expression of proteins in patients with various diseases through gene regulation. As small RNA has an increasing impact on biological pathways and changes the biological status of many genes, the extraction and separation of small RNA has attracted more and more attention from researchers in recent years.

[0003] The extraction method and process standardization of small RNA are key factors to ensure the quality of small RNA. High-purity and high-yield small RNA extraction methods or kits help ensure high-quality and accurate downstream research applications. In recent years, many researchers have conducted research based on small RNA extraction methods. There are various small RNA extraction methods, and the commonly used methods are precipitation method and filter column method. The precipitation method mainly uses the TRIZOL extraction method to directly extract small RNA from cells or tissues. Trizol contains phenol, guanidine isothiocyanate and other substances, 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. In addition, this method has low extraction efficiency, large sample volume requirements, and organic reagent residues will inhibit subsequent PCR reactions; the filter column method mainly uses centrifugation to extract small RNA, and uses the principle of high salt adsorption and low salt elution to extract small RNA. However, small RNA fragments are widely distributed, about 15-200bp, and are not easily adsorbed and precipitated. In addition, this method usually causes the loss of small RNA and destroys the integrity of RNA.

[0004] Currently, there is no relatively fast and efficient method for extracting small RNA. Therefore, there is an urgent need in the art to develop an efficient, accurate and high-purity method for extracting small RNA from biological fluids. Summary of the invention

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

[0006] The first aspect of the present invention provides a reagent for extracting small RNA, the reagent comprising a lysate, the lysate comprising 1-10 M guanidine isothiocyanate, 0.1-1 M ammonium thiocyanate, and 50-300 mM Tris; The reagent also includes a washing solution, which includes 5-50 mM Tris, 5-50 mM NaCl and 65%-75% anhydrous ethanol.

[0007] Preferably, the concentration of the guanidine isothiocyanate is 4-6 M, the concentration of the ammonium thiocyanate is 0.1-0.5 M, and the concentration of Tris is 50-200 mM.

[0008] Preferably, the pH of the lysate is 6.5-7.5.

[0009] Preferably, the reagent comprises an adsorption solution, 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.

[0010] Preferably, the SiC adsorption beads have a particle size of 4-10 μm and a purity greater than 95%.

[0011] Preferably, the NaCl concentration in the rinsing solution is 10-50 mM, and the volume fraction of the anhydrous ethanol is 70-75%.

[0012] The second aspect of the present invention provides a kit comprising any one of the reagents described above.

[0013] The third aspect of the present invention provides a method for extracting small RNA using any of the reagents described above, the method comprising 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.

[0014] Preferably, in step (1), the volume ratio of the sample to the lysate is 1:(1.8-2.2).

[0015] Preferably, the volume ratio of the sample to the adsorption liquid is 1:(0.05-0.2).

[0016] Preferably, the incubation temperature is 50-65° C., and the incubation time is 5-10 min.

[0017] Preferably, the sample is selected from the group consisting of 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.

[0018] Preferably, in step (2), the volume of the anhydrous ethanol is 2-4 times the volume of the sample.

[0019] Preferably, 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).

[0020] Preferably, in step (4), the elution solution is DEPC water or nuclease-free water.

[0021] Preferably, in step (4), the volume of the eluent is 30-100 μL.

[0022] The fourth aspect of the present invention provides use of any of the above reagents in extracting small RNA.

[0023] Compared with the prior art, the present invention has the following technical effects: The reagent for extracting small RNA provided by the present invention includes a lysis solution with specific components, so that the lysis solution can fully and effectively lyse cells with high lysis efficiency. With the cooperation of other reagents, small RNA in the sample can be quickly and efficiently obtained, and the obtained small RNA has high purity and high yield of small RNA, which is convenient for subsequent experiments (such as RT-qPCR, second-generation sequencing library construction, etc.); and the extraction method provided by the present invention has the advantages of simple operation, low cost, green and safe, etc., and has broad application prospects and huge market promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The quality test results of small RNA extracted by Qiagen kit in Example 1 of the present invention; Figure 2 The quality test result of small RNA extracted by the method provided by the present invention in Example 1 of the present invention; Figure 3 The quality test results of small RNA extracted by Qiagen kit in Example 2 of the present invention; Figure 4 The quality test result of small RNA extracted by the method provided by the present invention in Example 2 of the present invention; Figure 5 The peak diagram of constructing a second-generation sequencing library after extracting small RNA using the Qiagen kit in Example 2 of the present invention; Figure 6 A peak diagram of constructing a next-generation sequencing library after extracting small RNA using the method provided by the present invention in Example 2 of the present invention; Figure 7 The quality test results of small RNA extracted by Qiagen kit in Example 3 of the present invention; Figure 8 The quality test result of small RNA extracted by the method provided by the present invention in Example 3 of the present invention; Fig. 9 This is a bar graph comparing CT after RT-qPCR after small RNA was extracted using the Qiagen kit and the method provided by the present invention in Example 3; Fig.10 The quality test results of small RNA extracted by Qiagen kit in Example 4 of the present invention; Fig.11 The quality test result of small RNA extracted by the method provided by the present invention in Example 4 of the present invention; Fig.12 The quality test results of small RNA extracted by the Novozyme kit in Example 4 of the present invention; Fig.13 This is the quality test result of small RNA extracted by the method provided by the present invention in Example 4 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0026] High-quality RNA is the first and key step in molecular biology research, which directly affects the success or failure of the experiment. With the increasing clinical application of small RNA in biological fluids (such as plasma or serum), high-quality and high-concentration methods for extracting small RNA in biological fluids are monopolized by foreign countries, which has affected the development of small RNA research in biological fluids in my country. Based on this, the inventors proposed a method for extracting small RNA from biological fluids. After the method forms a kit, it can be used to extract small RNA from biological fluids such as cells, serum, plasma, exosomes, and urine. The product extracted by the kit can be directly applied to the detection of small RNA, such as RT-qPCR, second-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 extracted nucleic acid fragments.

[0027] In a first aspect, the present invention provides a reagent for extracting small RNA, the reagent comprising a lysate, the lysate comprising 1-10 M (e.g., 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 (e.g., 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, 50-300 mM (e.g., 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, 145 280 mM mM, 290 mM, 300 mM, etc.) Tris; The reagent also includes a rinse solution, and the rinse solution includes 5-50mM (e.g., 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, 5-50mM (such as 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, etc. mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50 mM, etc.) NaCl and 65%-75% (e.g., 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, etc.) anhydrous ethanol.

[0028] In certain embodiments, the lysate consists of 1-10 M guanidine isothiocyanate, 0.1-1 M ammonium thiocyanate, and 50-300 mM Tris.

[0029] In certain embodiments, the concentration of the guanidine isothiocyanate is 4-6 M, the concentration of the ammonium thiocyanate is 0.1-0.5 M, and the concentration of Tris is 50-200 mM.

[0030] In certain embodiments, the pH of the lysate is 6.5-7.5.

[0031] In certain embodiments, the pH of the lysate is 6.9-7.5.

[0032] In some embodiments, the reagent includes an adsorption liquid, and the adsorption liquid is composed of SiC adsorption beads and the lysate, and the concentration of the SiC adsorption beads is 0.2-1g / mL. The present application uses a lysate to fix the volume of SiC adsorption beads to form an adsorption liquid, which can more fully combine and extract the nucleic acid released in the supernatant to further improve the nucleic acid extraction rate and purity of the sample. At the same time, it saves costs and does not need to use other reagents as adsorption liquids.

[0033] In certain embodiments, the SiC adsorption beads have a particle size of 4-10 μm and a purity of greater than 95%.

[0034] In certain embodiments, the rinse solution consists of 5-50 mM Tris, 5-50 mM NaCl, and 65%-75% anhydrous ethanol.

[0035] In the present application, 65%-75% anhydrous ethanol refers to anhydrous ethanol with a volume fraction of 65%-75%.

[0036] In certain embodiments, the concentration of NaCl in the rinse solution is 10-50 mM, and the concentration of anhydrous ethanol is 70-75%.

[0037] In a second aspect, the present invention provides a kit of any of the reagents described above.

[0038] In a third aspect, the present invention provides a method for extracting small RNA using any of the reagents described above, the method comprising 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.

[0039] In certain embodiments, in step (1), the volume ratio of the sample to the lysate is 1:(1.8-2.2), for example, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2.

[0040] In some embodiments, the volume ratio of the sample to the adsorption liquid is 1:(0.05-0.2), for example, 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.

[0041] In certain embodiments, the incubation temperature is 50-65°C (e.g., 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 incubation time is 5-10 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.).

[0042] In certain embodiments, the sample is selected from the group consisting of 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, ocular fluid, amniotic fluid, placental fluid, ascites, pleural effusion, sputum, bronchial aspirate, and exosomes.

[0043] In certain embodiments, the sample is selected from the group consisting of blood, serum, saliva, urine, plasma, and exosomes.

[0044] In certain embodiments, in step (2), the volume of the anhydrous ethanol is 2-4 times, for example, 2 times, 2.5 times, 3 times, 3.5 times, or 4 times, the volume of the sample.

[0045] In certain embodiments, 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).

[0046] In some embodiments, step (3) can be repeated multiple times (e.g., 1 time, 2 times, 3 times, 4 times, 5 times). In a preferred embodiment, step (3) is repeated 2 times.

[0047] In certain embodiments, in step (4), the elution solution is DEPC water or nuclease-free water.

[0048] In certain embodiments, in step (4), the volume of the eluent is 30-100 μL, for example, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, etc.

[0049] In certain embodiments, the lysate comprises 1-10 M (e.g., 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 (e.g., 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, 50-300 mM (e.g., 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, 155 300 mM mM, etc.) Tris.

[0050] In certain embodiments, the concentration of guanidine thiocyanate is 4-6 M guanidine thiocyanate, 0.1-0.5 M ammonium thiocyanate, and 50-200 mM Tris.

[0051] In certain embodiments, the pH of the lysate is 6.5-7.5.

[0052] 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.

[0053] In certain embodiments, the SiC adsorption beads have a particle size of 4-10 μm and a purity of greater than 95%.

[0054] In certain embodiments, the rinse solution comprises 5-50 mM (e.g., 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, 5-50mM (such as 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, etc. mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50 mM, etc.) NaCl and 65%-75% (e.g., 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, etc.) anhydrous ethanol.

[0055] In certain embodiments, the NaCl concentration in the rinse solution is 10-50 mM, and the volume fraction of the anhydrous ethanol is 70-75%.

[0056] The fourth aspect of the present invention provides use of any of the above reagents in extracting small RNA.

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sam Brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts. Example 1

[0058] Reagent preparation in this example: (1) Lysis solution (lysis buffer): 5 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0; (2) Adsorption solution: The lysis solution is used to adjust the volume of adsorption beads (SiC) to a mass concentration of 1 g / mL, wherein the particle size of the adsorption beads is 5 μm and the purity is 95.5%; (3) Rinsing solution: 15 mM Tris, 15 mM NaCl, 73% anhydrous ethanol.

[0059] The steps of extracting small RNA from a whole blood sample using the above reagents include: (1) Mix the whole blood sample and centrifuge it. Add 1.9 times the volume of lysis solution and 0.1 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 55°C for 5 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0060] At the same time, in this example, Qiagen miRNA extraction kit (Qiagen, miRNeasy Serum / Plasma Advanced Kit, catalog number: 217204) was used to extract small RNA for control.

[0061] The extracted product was tested for quality using an Agilent small RNA kit. The quality test results of small RNA extracted with the Qiagen kit are as follows: Figure 1 As shown, the quality test results of small RNA extracted by the method provided by the present invention are as follows Figure 2 As shown, Figure 1By comparison, with the 4 bp peak as a reference, it can be seen that the method provided by the present invention has a higher concentration of 20-40 bp nucleic acid products, accounting for a larger proportion. Therefore, the product obtained by the method provided by the present invention has a higher concentration, and there is an obvious peak after 150 bp, indicating that the abundance of small RNA is better. Example 2

[0062] Reagent preparation in this example: (1) Lysis solution (lysis buffer): 5.5 M guanidine isothiocyanate, 0.3 M ammonium thiocyanate, 50 mM Tris, pH = 6.9; (2) Adsorption solution: Use the lysis solution to make up the volume of adsorption beads (SiC) until the mass concentration of SiC is 0.75 g / mL, where the particle size of the adsorption beads is 5 μm and the purity is 95%; (3) Rinsing solution: 25 mM Tris, 25 mM NaCl, 71% anhydrous ethanol.

[0063] The steps of extracting small RNA from plasma samples using the above reagents include: (1) Mix the plasma sample and centrifuge it. Add 1.8 times the volume of lysis solution and 0.2 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 56°C for 10 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 50 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0064] At the same time, in this example, Qiagen serum / plasma miRNA extraction kit (Qiagen, miRNeasy Serum / Plasma Advanced Kit, catalog number: 217204) was used to extract small RNA for control.

[0065] The extracted products were quality tested using an Agilent small RNA kit, and downstream second-generation sequencing libraries were constructed using a miRNA library construction kit (Qiagen, QIAseq miRNA Library Kit). The library construction and quality inspection methods are described in the instructions for the Qiagen miRNA library construction kit (Qiagen, QIAseq miRNA Library Kit). The quality inspection results of small RNA extracted by the Qiagen kit are shown in Figure 3 As shown, the quality test results of small RNA extracted by the method provided by the present invention are as follows Figure 4 As shown, through Figure 3 Compared with the 4 bp peak, it can be seen that the 20-40 bp nucleic acid concentration obtained by the method provided by the present invention is higher and accounts for a larger proportion; the peak diagram of the second-generation sequencing library constructed after the Qiagen kit extracted small RNA is shown in Figure 5 The peak diagram of constructing the second generation sequencing library after extracting small RNA by the method provided by the present invention is shown in Figure 6 ,from Figure 6 It can be seen that the product obtained by the method provided by the present invention is of higher quality, and the small RNA obtained by the method provided by the present invention can be directly applied to the construction of the second-generation sequencing library. Example 3

[0066] Reagent preparation in this example: (1) Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 55 mM Tris, pH = 7.1; (2) Adsorption solution: Use the lysis solution to make up the volume of adsorption beads (SiC) until the mass concentration of SiC is 1 g / mL, where the particle size of the adsorption beads is 7.5 μm and the purity is 95%; (3) Rinsing solution: 25 mM Tris, 25 mM NaCl, 75% anhydrous ethanol.

[0067] The steps of extracting small RNA from a serum sample using the above reagent include: (1) Add 5 μL of 50 nM cel-39 (purchased from Qiagen) per milliliter of serum, mix well and briefly centrifuge. Add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 56°C for 10 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0068] Meanwhile, in this example, Qiagen miRNeasy Serum / Plasma Advanced Kit was used to extract small RNA for control.

[0069] The extracted products were tested for quality using an Agilent small RNA kit, and several small RNAs (Cel-39, miR-142-3P, miR-26b) were selected and compared in concentration using the Thermo Fisher qPCR method. The miRNA reverse transcription step was performed according to the instructions of the kit (TaqMan MicroRNA Reverse Transcription Kit, Catalog No.: 4366596), the pre-amplification method was performed according to the Thermo Fisher pre-amplification kit (TaqMan PreAmp Master Mix, Catalog No.: 4384266), and the Thermo Fisher qPCR kit (TaqMan Fast Advanced Master Mix, Catalog No.: 4444557) was used for real-time quantitative PCR. The results are shown in Figures 7 to 9 .

[0070] The quality test results of small RNA extracted by Qiagen kit are as follows Figure 7 As shown, the quality test results of small RNA extracted by the method provided by the present invention are as follows Figure 8 As shown, Figure 7 By comparison, with the 4bp peak as a reference, it can be seen that the method provided by the present invention has a higher concentration of 20-40bp nucleic acid products, accounting for a larger proportion, so the method provided by the present invention has a higher concentration of products. In addition, there is an obvious peak after 150bp, indicating that the abundance of small RNA is better.

[0071] Fig. 9The CT bar graphs of RT-qPCR after small RNA was extracted using the Qiagen kit and the method provided by the present invention, wherein Q1 and Q2 are CT bar graphs of RT-qPCR after RNA was extracted using the Qiagen kit, and ZY1 and ZY2 are CT bar graphs of RT-qPCR after RNA was extracted using the kit of the present invention. From the CT value, the CT of the kit of the present invention is smaller and better. Example 4

[0072] Reagent preparation in this example: (1) Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 55 mM Tris, pH = 7.0; (2) Adsorption solution: Use the lysis solution to make up the volume of adsorption beads (SiC) until the mass concentration of SiC is 1 g / mL, where the particle size of the adsorption beads is 5 μm and the purity is 95%; (3) Rinsing solution: 25 mM Tris, 25 mM NaCl, 73% anhydrous ethanol.

[0073] The steps of extracting small RNA from exosome samples using the above reagents include: (1) Plasma exosomes were extracted using the MARCKS-ED magnetic bead exosome enrichment and purification kit (reference CN115558629A) from Ehang Biotech (Suzhou); (2) Based on the sample volume, add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution to the sample to obtain a mixed solution 1, and incubate at 56°C for 10 min; (3) adding 3 times the sample volume of anhydrous ethanol to the mixed solution 1, briefly centrifuging, and discarding the supernatant to obtain a mixture 2; (4) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (5) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (6) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (7) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (8) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (9) Detection of small RNAs.

[0074] Meanwhile, in this example, Qiagen miRNeasy Serum / Plasma Advanced Kit was used to extract small RNA for control.

[0075] The extracted product was tested for quality using an Agilent small RNA kit. The quality test results of small RNA extracted with the Qiagen kit are as follows: Fig.10 As shown, the quality test results of small RNA extracted by the method provided by the present invention are as follows Fig.11 As shown, from Fig.10 In comparison, it can be seen that the product obtained by the method provided by the present invention has a higher concentration and an obvious peak after 150 bp, indicating that the abundance of small RNA is better ( Fig.11 ). Example 5

[0076] Reagent preparation in this example: (1) Lysis solution (lysis buffer): 5 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0; (2) Adsorption solution: Use the lysis solution to make up the volume of adsorption beads (SiC) until the mass concentration of SiC is 1 g / mL, where the particle size of the adsorption beads is 7.5 μm and the purity is 95%; (3) Rinse solution: 50 mM Tris, 50 mM NaCl, 75% anhydrous ethanol.

[0077] The steps of extracting small RNA from urine samples using the above reagents include: (1) Mix the urine sample and centrifuge it. Add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 55°C for 10 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0078] Meanwhile, in this example, the Trizol method was used to extract RNA, and the reagents used were purchased from Novozymes (Cat. No.: R401-01) as a control.

[0079] The extracted product was tested for quality using an Agilent small RNA kit. The quality test results of the small RNA extracted by the Novozyme kit are as follows: Fig.12 As shown, the quality test results of small RNA extracted by the method provided by the present invention are as follows Fig.13 As shown, Fig.12 In comparison, it can be seen that the product concentration obtained by the method provided by the present invention is higher. Example 6

[0080] In this example, other lysate formulas are selected to be used in combination with the adsorption solution and the cleaning solution of the present invention to extract small RNA. The specific lysate formula is shown in Table 1: Table 1

[0081] Adsorption solution: Use lysis solution to make up the volume of adsorption beads (SiC) to a mass concentration of 1g / mL, where the particle size of the adsorption beads is 7.5μm and the purity is 95%; Rinsing solution: 50mM Tris, 50mM NaCl, 75% anhydrous ethanol.

[0082] The above reagents were used to extract small RNA from plasma and serum samples, respectively, and the operation steps were as follows: (1) Add 5 μL of 50 nM cel-39 (purchased from Qiagen) per milliliter of serum or plasma, mix well and briefly centrifuge. Add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 55°C for 10 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0083] The extracted products were tested at 2100 and the concentration of small RNA was calculated. Several small RNAs (U6, Cel-39, miR-26b) were selected from the extracted products and the concentrations were compared using the Thermo Fisher reagent qPCR method, the steps of which are described in Example 3. The test results of small RNA extracted from different samples using different lysis buffers are shown in Table 2: Table 2

[0084] The results in Table 2 show that the concentration of small RNA extracted by the reagent provided by the present invention (Groups 1 and 2) is the highest, and the CT value after superposition is smaller, indicating that the reagent of the present invention has the best effect. Example 7

[0085] In this example, other rinse solution formulas are selected to be used in combination with the lysate and adsorption solution of the present invention to extract small RNA. The specific rinse solution formula is shown in Table 3: Table 3

[0086] Lysis solution (lysis buffer): 5.0 M guanidine isothiocyanate, 0.2 M ammonium thiocyanate, 100 mM Tris, pH = 7.0; Adsorption solution: Use lysis solution to make up the volume of adsorption beads (SiC) to a mass concentration of 1g / mL, where the particle size of the adsorption beads is 7.5μm and the purity is 95%; The above reagents were used to extract small RNA from plasma and serum samples, respectively, and the operation steps were as follows: (1) Add 5 μL of 50 nM cel-39 (purchased from Qiagen) per milliliter of serum or plasma, mix well and briefly centrifuge. Add 1.9 times the volume of lysis buffer and 0.1 times the volume of adsorption solution based on the sample volume to obtain mixed solution 1, and incubate at 55°C for 10 min. (2) adding 3 times the volume of the sample to the mixed solution 1 with anhydrous ethanol, centrifuging briefly, and discarding the supernatant to obtain a mixture 2; (3) adding 350 μL of lysate to the mixture 2, and then adding 350 μL of anhydrous ethanol, and mixing to obtain a mixture 3; (4) Transfer the mixed solution 3 to a silica membrane column (purchased from Hangzhou Xinjing) and centrifuge at 12000g for 1 min; (5) Add 500 μL of rinse solution to the silica membrane column and centrifuge at 12,000 g for 1 min. Repeat twice. (6) Add 100 μL of DEPC water to the silica membrane column and let it stand for 2 min; (7) Collect the eluate in a nuclease-free centrifuge tube and centrifuge at 13,000 g for 2 min; (8) Detection of small RNAs.

[0087] The extracted products were tested at 2100 and the concentration of small RNA was calculated. Several small RNAs (U6, Cel-39, miR-26b) were selected from the extracted products and the concentrations were compared using the Thermo Fisher reagent qPCR method, the steps of which are described in Example 3. The test results of extracting small RNAs from different samples using a kit composed of different rinse solutions and the lysis solution and adsorption solution of the present invention are shown in Table 4: Table 4

[0088] It can be seen from Table 4 that, regardless of whether the sample is plasma or serum, the concentration of small RNA extracted using the reagent provided by the present invention (Groups 7 and 8) is the highest, and the CT value after superposition is smaller, indicating that the reagent of the present invention has the best effect and a wide range of applicability to samples.

[0089] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A reagent for extracting small RNA, characterized in that: 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; The reagent also includes a washing solution, which includes 5-50 mM Tris, 5-50 mM NaCl and 65%-75% anhydrous ethanol.

2. The reagent according to claim 1, characterized in that The concentration of the guanidine isothiocyanate is 4-6 M, the concentration of the ammonium thiocyanate is 0.1-0.5 M, and the concentration of the Tris is 50-200 mM; And / or, 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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