Chloroform-free extraction RNA lysis buffer as well as preparation method and application thereof

Abstract

The invention provides a chloroform-free extraction RNA lysis buffer as well as a preparation method and application thereof, and relates to the technical field of biology. Comprising the following components in concentration: 1-5 mol / L chaotropic salt, 0.1%-5% (v / v) of an anionic surfactant, 0.01-50 mmol / L of an ionic chelating agent, 0.01%-50% (v / v) of a phenol solution, 30-300 mmol of buffer salt, 0.01%-1% (v / v) of a reducing agent, 0.2%-1% (w / v) of a cosolvent, 10%-20% (w / v) of a separating agent and 0.01%-0.2% (w / v) of a fat-soluble indicator. According to the method disclosed by the invention, the use of volatile highly toxic organic reagents such as chloroform can be avoided, and the quality of the extracted RNA is superior to that of the traditional RNA lysate. The technical problems that in the prior art, lysate needs to depend on volatile highly toxic organic reagents such as chloroform and the like, and the extraction time is long are solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a chloroform-free RNA extraction lysis solution and a preparation method and application thereof. Background Art

[0002] RNA is a type of molecule that is easily degraded. To obtain complete RNA, it is necessary to minimize the degradation of RNA by endogenous and exogenous ribonucleases during the extraction process. High-concentration, strongly denaturing chaotropic salts can quickly dissolve proteins, destroy cell structures, and separate nucleoproteins from nucleic acids; at the same time, they can inhibit nucleases and RNAases released by cells, protecting RNA from degradation when released from cells. The products released after cell lysis contain not only RNA, but also substances such as DNA, proteins, and cell fragments. At this time, the cell lysis products can be treated with organic agents such as chloroform to obtain purified and uniform total RNA.

[0003] At present, RNA lysis solutions from major manufacturers at home and abroad are mainly based on the use of chloroform or other organic compounds to extract and purify various types of samples. However, chloroform and other organic reagents are highly toxic and volatile, most of which are controlled compounds that are difficult to obtain, and the extraction time is long, and RNA is more easily degraded during the extraction process.

[0004] Existing RNA lysing solutions are mainly based on the extraction and purification of various types of samples by combining with chloroform or other organic compounds. For example, the Chinese patent with application number 201210380436.9 discloses a reagent and method for quickly extracting total RNA from a liquid sample, the main components of which are guanidine isothiocyanate, acetic acid, phenol, ammonium thiocyanate, sodium lauryl sarcosine, and sodium acetate. The disclosed components are all classic compounds of conventional RNA lysing solutions, and the experiment requires the use of chloroform reagents to extract and purify the samples. The Chinese patent with application number 202410961585.7 discloses a chloroform-free and phase-free rapid RNA extraction method based on TRIZOL lysing solution and its application, which mainly consists of TRIZOL reagent, TRIZOL binding solution, RNA rinse solution 1, and RNA rinse solution 2. The inventor improved the extraction process based on the use of TRIZOL reagent, but the core component of the sample lysing treatment, TRIZOL reagent, is a commercially available product, and its innovation only makes it simpler and faster than the original operation scheme. The Chinese patent with application number 202011378567.4 discloses a nucleic acid dissociation solution and its application in nucleic acid extraction, the main components of which are guanidine isothiocyanate, water-saturated phenol, alcohol reducing agent and 8-hydroxyquinoline. The inventor relies on dextran gel technology to quickly purify RNA, but the data displayed only show that it is used in PCR detection, and the purity and integrity data of the obtained RNA are not visible. Therefore, the compatibility of the invention with downstream applications is unknown. The Chinese patent with application number 202310929960.5 discloses an RNA extraction method, the main components of which are phenol, protein denaturant, detergent, buffer salt, disulfide bond reducing agent, pH regulator, etc. Its disclosed components are all classic compounds of conventional RNA lysis solution, and the experiment needs to rely on the use of chloroform reagent to extract and purify the sample. At the same time, because the organic reagents used in RNA lysis, extraction and purification are highly toxic and volatile, they are easy to endanger the safety of experimenters.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a chloroform-free RNA lysis solution to at least solve one of the technical problems existing in the prior art.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned chloroform-free RNA extraction lysate.

[0008] The third object of the present invention is to provide the use of the chloroform-free RNA extraction lysate or the chloroform-free RNA extraction lysate obtained by the above-mentioned preparation method in RNA extraction.

[0009] A fourth object of the present invention is to provide a kit for extracting RNA without chloroform.

[0010] A fifth object of the present invention is to provide a method for extracting RNA without chloroform.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a chloroform-free RNA extraction lysis solution, comprising the following components in concentrations: 1 to 5 mol / L of a chaotropic salt, 0.1% to 5% (v / v) of an anionic surfactant, 0.01 to 50 mmol / L of an ion chelating agent, 0.01% to 50% (v / v) of a phenol solution, 30 to 300 mmol of a buffer salt, 0.01% to 1% (v / v) of a reducing agent, 0.2% to 1% (w / v) of a cosolvent, 10% to 20% (w / v) of a separating agent, and 0.01% to 0.2% (w / v) of a fat-soluble indicator.

[0013] Further, the chaotropic salt includes at least one of guanidine thiocyanate, ammonium thiocyanate, guanidine hydrochloride, sodium perchlorate, sodium periodate or sodium iodide;

[0014] Preferably, the anionic surfactant includes at least one of sodium lauryl sarcosinate, sodium lauryl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate or dodecyl triethanolamine;

[0015] Preferably, the ion chelating agent comprises at least one of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid or 8-hydroxyquinoline;

[0016] Preferably, the phenol solution comprises at least one of water-saturated phenol, citric acid-saturated phenol or phenol;

[0017] Preferably, the buffer salt comprises at least one of tris(hydroxymethylaminomethane), sodium citrate, sodium acetate or potassium acetate;

[0018] Preferably, the reducing agent comprises at least one of β-mercaptoethanol, dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride or reduced glutathione;

[0019] Preferably, the cosolvent comprises at least one of dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran;

[0020] Preferably, the separating agent comprises at least one of hexamethylphosphoramide or tetramethylethylenediamine;

[0021] Preferably, the fat-soluble indicator comprises at least one of solvent blue 36, solvent blue 38, bromocresol green or phenol red.

[0022] Furthermore, the pH of the chloroform-free RNA extraction lysis solution is 3-5.

[0023] In a second aspect, the present invention provides a method for preparing the above-mentioned chloroform-free RNA extraction lysate, which is prepared by dissolving each component in water according to the formula amount.

[0024] In a third aspect, the present invention provides the use of the chloroform-free RNA extraction lysate or the chloroform-free RNA extraction lysate obtained by the above-mentioned preparation method in RNA extraction.

[0025] In a fourth aspect, the present invention provides a kit for chloroform-free RNA extraction, comprising the above-mentioned chloroform-free RNA extraction lysate or the chloroform-free RNA extraction lysate prepared by the above-mentioned preparation method.

[0026] Furthermore, the kit also includes an RNA extraction solution;

[0027] Preferably, the RNA extraction solution comprises isopropanol;

[0028] Preferably, the kit further comprises an RNA elution solution;

[0029] Preferably, the RNA elution solution comprises an alcohol solution or water.

[0030] In a fifth aspect, the present invention provides a method for extracting RNA without chloroform, the method comprising the following steps:

[0031] A. adding the chloroform-free RNA extraction lysis solution or the chloroform-free RNA extraction lysis solution prepared by the above-mentioned preparation method to the sample cell suspension, shaking and dissolving, then standing, and centrifuging to obtain a lysis product;

[0032] B. Mix the RNA extract and the lysate, incubate, and centrifuge to obtain RNA precipitate.

[0033] Furthermore, the method for preparing the sample cell suspension includes taking a cell suspension, centrifuging to obtain a precipitate, and adding water to resuspend the precipitate to obtain a sample cell suspension;

[0034] Preferably, the cell amount of the sample cell suspension is less than 5×10 7 ;

[0035] Preferably, the amount of the chloroform-free RNA extraction lysis solution used is 1-2 mL of chloroform-free RNA extraction lysis solution per 100 μL of sample cell suspension, preferably 1 mL;

[0036] Preferably, the RNA extract and the lysate are mixed in equal volumes;

[0037] Preferably, the standing time in step A is 3 to 10 minutes;

[0038] Preferably, the centrifugal speed in step A is 10000-14000 rpm, preferably 12000 rpm;

[0039] Preferably, the centrifugation time in step A is 12 to 18 minutes, preferably 15 minutes;

[0040] Preferably, the centrifugation temperature in step A is 0-6°C, preferably 4°C;

[0041] Preferably, the incubation temperature in step B is 25-37°C;

[0042] Preferably, the incubation time in step B is 3 to 10 minutes;

[0043] Preferably, the centrifugal speed in step B is 10000-14000 rpm, preferably 12000 rpm;

[0044] Preferably, the centrifugation time in step B is 8 to 15 minutes, preferably 10 minutes;

[0045] Furthermore, the method further comprises step C, dispersing the RNA precipitate in an RNA elution solution, and centrifuging to obtain the RNA precipitate;

[0046] Preferably, the amount of the RNA elution solution is 1-2 mL of RNA elution solution per 100 μL of sample cell suspension, preferably 1 mL;

[0047] Preferably, the operation of step C is repeated 1 to 2 times;

[0048] Preferably, after step C, the method further comprises dissolving the dried RNA precipitate in water and incubating to obtain an RNA solution;

[0049] Preferably, the water used to dissolve the RNA precipitate is added in equal volumes as the sample cell suspension;

[0050] Preferably, the incubation temperature in step C is 60-70°C, preferably 65°C;

[0051] Preferably, the incubation time in step C is 2 to 5 minutes.

[0052] The present invention provides a chloroform-free RNA extraction lysis solution, which increases the solubility between the organic reagent and the aqueous solution by using a cosolvent to stabilize the mixed solution; a separation agent is added to extract and purify the sample RNA without adding volatile toxic organic reagents such as chloroform or chloroform substitutes. Compared with the traditional RNA lysis solution that relies on volatile and highly toxic organic reagents such as chloroform to extract and purify the sample, the present invention can get rid of the use of the above-mentioned volatile and highly toxic organic reagents, and the quality of the extracted RNA is better than that of the traditional RNA lysis solution. It solves the technical problem that the lysis solution in the prior art needs to rely on volatile and highly toxic organic reagents such as chloroform, and the extraction time is long. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 Agilent 4150 test results of the RNA solution provided in Experimental Example 1 of the present invention;

[0055] Figure 2 Agilent 4150 test results of the RNA solution provided in Experimental Example 21 of the present invention. DETAILED DESCRIPTION

[0056] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0057] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0058] On one hand, the present invention provides a chloroform-free RNA lysis solution, which comprises the following components in concentrations: 1-5 mol / L of a chaotropic salt, 0.1%-5% (v / v) of an anionic surfactant, 0.01-50 mmol / L of an ion chelating agent, 0.01%-50% (v / v) of a phenol solution, 30-300 mmol of a buffer salt, 0.01%-1% (v / v) of a reducing agent, 0.2%-1% (w / v) of a cosolvent, 10%-20% (w / v) of a separating agent and 0.01%-0.2% (w / v) of a fat-soluble indicator.

[0059] By using a co-solvent to increase the solubility between the organic reagent and the aqueous solution, the mixed solution is kept stable; a separation agent is added to extract and purify the sample RNA without adding volatile toxic organic reagents such as chloroform or chloroform substitutes. Compared with the traditional RNA lysing solution that relies on volatile and highly toxic organic reagents such as chloroform to extract and purify the sample, the present invention can get rid of the use of the above-mentioned volatile and highly toxic organic reagents, and the quality of the extracted RNA is better than that of the traditional RNA lysing solution. It solves the technical problem that the lysing solution of the prior art needs to rely on volatile and highly toxic organic reagents such as chloroform, and the extraction time is long.

[0060] The concentration of the chaotropic salt may be, but is not limited to, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L or 5 mol / L, or any concentration between 1 and 5 mol / L.

[0061] The concentration of the anionic surfactant may be, but is not limited to, 0.1% (v / v), 0.5% (v / v), 1% (v / v), 1.5% (v / v), 2% (v / v), 2.5% (v / v), 3% (v / v), 3.5% (v / v), 4% (v / v), 4.5% (v / v) or 5% (v / v), and may be any concentration between 0.1% and 5% (v / v).

[0062] The concentration of the ion chelator may be, but is not limited to, 0.01 mmol / L, 0.05 mmol / L, 1 mmol / L, 3 mmol / L, 5 mmol / L, 8 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L or 50 mmol / L, and may be any concentration between 0.01 and 50 mmol / L.

[0063] The concentration of the phenol solution may be, but is not limited to, 0.01%, 0.1%, 1%, 3%, 5%, 8%, 10%, 20%, 30%, 40% or 50%, or any concentration between 0.01% and 50% (v / v).

[0064] The concentration of the buffer salt may be, but is not limited to, 30 mmol, 50 mmol, 80 mmol, 100 mmol, 130 mmol, 150 mmol, 180 mmol, 200 mmol, 230 mmol, 250 mmol, 280 mmol or 300 mmol, or any concentration between 30 and 300 mmol.

[0065] The concentration of the reducing agent may be, but is not limited to, 0.01%, 0.03%, 0.05%, 0.08%, 0.1%, 0.3%, 0.5%, 0.8% or 1%, and may be any concentration between 0.01% and 1% (v / v).

[0066] The concentration of the cosolvent may be, but is not limited to, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, or any concentration between 0.2% and 1% (w / v).

[0067] The concentration of the separating agent may be, but is not limited to, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, and may be any concentration between 10% and 20% (w / v).

[0068] The concentration of the fat-soluble indicator may be, but is not limited to, 0.01%, 0.05%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, or any concentration between 0.01% and 0.2% (w / v).

[0069] In some specific embodiments, the chaotropic salt includes at least one of guanidine thiocyanate, ammonium thiocyanate, guanidine hydrochloride, sodium perchlorate, sodium periodate or sodium iodide. In some specific embodiments, the anionic surfactant includes at least one of sodium lauryl sarcosinate, sodium lauryl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate or dodecyl triethanolamine. In some specific embodiments, the ion chelator includes at least one of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, ethylene glycol bis (2-aminoethyl ether) tetraacetic acid or 8-hydroxyquinoline; in some specific embodiments, the phenol solution includes at least one of water-saturated phenol, citric acid saturated or phenol; in some specific embodiments, the buffer salt includes at least one of trishydroxymethylaminomethane, sodium citrate, sodium acetate or potassium acetate; in some specific embodiments, the reducing agent includes at least one of β-mercaptoethanol, dithiothreitol, tris (2-carbonylethyl) phosphine hydrochloride or reduced glutathione; in some specific embodiments, the cosolvent includes at least one of dimethyl sulfoxide, N, N-dimethylformamide or tetrahydrofuran; in some specific embodiments, the separating agent includes at least one of hexamethylphosphoramide or tetramethylethylenediamine. In some specific embodiments, the fat-soluble indicator includes at least one of solvent blue 36, solvent blue 38, bromocresol green or phenol red.

[0070] Here, "at least one" means selecting one or more from the listed ingredients, but is not limited to selecting only one, including a single ingredient or a combination of multiple ingredients. A single ingredient refers to the case of selecting a single ingredient from the listed ingredients, and a combination of multiple ingredients refers to a combination of two or more selected from the listed ingredients.

[0071] In some specific embodiments, the pH of the chloroform-free RNA extraction lysis solution is 3-5.

[0072] According to another aspect of the present invention, a method for preparing the above-mentioned chloroform-free RNA extraction lysis solution is also provided, which is prepared by dissolving each component in water according to the formula amount.

[0073] The water used for dissolution is enzyme-free water.

[0074] According to another aspect of the present invention, there is also provided the use of the chloroform-free RNA extraction lysate or the chloroform-free RNA extraction lysate obtained by the above preparation method in RNA extraction.

[0075] According to another aspect of the present invention, a kit for chloroform-free RNA extraction is provided, comprising the above-mentioned chloroform-free RNA extraction lysate or the chloroform-free RNA extraction lysate prepared by the above-mentioned preparation method.

[0076] In some specific embodiments, the kit further comprises an RNA extracting solution; in some specific embodiments, the RNA extracting solution comprises isopropanol.

[0077] In some specific embodiments, the kit further comprises an RNA elution solution; in some specific embodiments, the RNA elution solution comprises an alcohol solution or water.

[0078] According to another aspect of the present invention, a method for extracting RNA without chloroform is also provided, the method comprising the following steps:

[0079] A. Add the chloroform-free RNA extraction lysis solution or the chloroform-free RNA extraction lysis solution prepared by the above-mentioned preparation method to the sample cell suspension, shake until dissolved, let stand, and centrifuge to obtain a lysis product;

[0080] B. Mix the RNA extract and the lysate, incubate, and centrifuge to obtain RNA precipitate.

[0081] By using the above-mentioned chloroform-free RNA extraction lysis solution, it is achieved that sample RNA can be extracted and purified without relying on volatile and highly toxic organic reagents such as chloroform; and the quality of the extracted RNA is better than that of traditional RNA lysis solutions.

[0082] In some specific embodiments, the method for preparing the sample cell suspension includes taking a cell suspension, centrifuging to obtain a precipitate, and adding water to resuspend the precipitate to obtain a sample cell suspension.

[0083] In some specific embodiments, the cell amount of the sample cell suspension is less than 5×10 7 .

[0084] In some specific embodiments, the usage of the chloroform-free RNA extraction lysis solution is 1-2 mL of chloroform-free RNA extraction lysis solution per 100 μL of sample cell suspension.

[0085] The amount of the chloroform-free RNA extraction lysis solution used can be, but is not limited to, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL or 2 mL per 100 μL of sample cell suspension, or any volume between 1 and 2 mL, preferably 1 mL.

[0086] In some specific embodiments, the shaking includes manual vigorous shaking for 15 to 30 seconds.

[0087] In some specific embodiments, the RNA extraction solution and the lysis product are mixed in equal volumes.

[0088] In some specific embodiments, the standing time in step A is 3 to 10 minutes.

[0089] In some specific embodiments, the centrifugal speed in step A is 10000-14000 rpm, preferably 12000 rpm. In some specific embodiments, the centrifugal time in step A is 12-18 min, preferably 15 min. In some specific embodiments, the centrifugal temperature in step A is 0-6°C, preferably 4°C;

[0090] In some specific embodiments, the incubation temperature in step B is 25-37° C.; in some specific embodiments, the incubation time in step B is 3-10 min.

[0091] In some specific embodiments, the centrifugal speed in step B is 10000-14000 rpm, preferably 12000 rpm; in some specific embodiments, the centrifugal time in step B is 8-15 min, preferably 10 min.

[0092] In some specific embodiments, the method further comprises step C, dispersing the RNA precipitate in an RNA elution solution and centrifuging to obtain the RNA precipitate; in some specific embodiments, the amount of the RNA elution solution is 1 to 2 mL of RNA elution solution per 100 μL of sample cell suspension, preferably 1 mL.

[0093] In some specific embodiments, the operation of step C is repeated 1 to 2 times.

[0094] In some specific embodiments, the step C further comprises dissolving the dried RNA precipitate in water and incubating to obtain an RNA solution. In some specific embodiments, the water used to dissolve the RNA precipitate is added in equal volumes to the sample cell suspension.

[0095] In some specific embodiments, the incubation temperature in step C is 60-70° C., preferably 65; in some specific embodiments, the incubation time in step C is 2-5 min.

[0096] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0097] Example 1

[0098] A chloroform-free RNA extraction lysis solution comprises the following components: 2 mol guanidine thiocyanate, 0.5 mol ammonium thiocyanate, 2% (V / V) dodecyl triethanolamine, 0.01 mol 8-hydroxyquinoline, 50% (V / V) citric acid saturated phenol, 0.3 mol potassium acetate, 1% (V / V) beta-mercaptoethanol, 0.1% (W / V) N,N-dimethylformamide, 0.5% (W / V) hexamethylphosphoramide and 0.05% (W / V) phenol red.

[0099] Dissolve each component in enzyme-free water according to the above formula and adjust the volume to 1L to prepare chloroform-free RNA extraction lysis solution.

[0100] Example 2

[0101] The difference from Example 1 is that the reducing agent uses 50 mM dithiothreitol instead of 2% (V / V) β-mercaptoethanol, and the anionic surfactant uses 1% (V / V) sodium lauryl sarcosinate instead of 2% (V / V) dodecyl triethanolamine.

[0102] Example 3

[0103] The difference from Example 1 is that the reducing agent uses 20 mM tris(2-carbonylethyl)phosphine hydrochloride instead of 2% (V / V) β-mercaptoethanol, and the chaotropic salt uses 3 mol sodium perchlorate and 1.75 mol sodium periodate instead of 2 mol guanidine thiocyanate and 0.5 mol ammonium thiocyanate.

[0104] Example 4

[0105] The difference from Example 1 is that the reducing agent uses 2% (W / V) reduced glutathione instead of 2% (V / V) β-mercaptoethanol, and the phenol solution uses 50% (V / V) water-saturated phenol instead of 50% (V / V) citric acid-saturated phenol.

[0106] Comparative Example 1

[0107] The difference from Example 2 is that the co-solvent and the separating agent are not added.

[0108] Comparative Example 2

[0109] The difference from Example 2 is that the co-solvent is not added.

[0110] Comparative Example 3

[0111] The difference from Example 2 is that the separating agent is not added.

[0112] Comparative Example 4

[0113] The difference from Example 2 is that the concentration of guanidine thiocyanate is 6 mol / L.

[0114] Comparative Example 5

[0115] The difference from Example 2 is that the concentration of guanidine thiocyanate is 0.5 mol / L.

[0116] Comparative Example 6

[0117] The difference from Example 1 is that the concentration of the β-mercaptoethanol is 0.01% (V / V).

[0118] Comparative Example 7

[0119] The difference from Example 1 is that the concentration of the β-mercaptoethanol is 0.1% (V / V).

[0120] Embodiments 5 to 8

[0121] A chloroform-free RNA extraction kit is provided, wherein the chloroform-free RNA extraction lysing solution prepared in Examples 1 to 4 is respectively selected as the RNA lysing solution in the kit. The kit specifically comprises an RNA lysing solution, an RNA extracting solution, and an RNA eluting solution. The RNA extracting solution is isopropanol; and the RNA eluting solution is 75% ethanol.

[0122] Comparative Examples 8 to 14

[0123] Different from Example 5, the chloroform-free RNA extraction lysis solutions prepared in Comparative Examples 1 to 7 are respectively selected as the RNA lysis solutions in this kit.

[0124] Comparative Example 15

[0125] Thermo Fisher TRIzol TM Kit (Cat. No. 15596026).

[0126] Experimental Example 1

[0127] 1. Sample: 1×10 6 The K562 cell suspension was used as the experimental sample for RNA extraction, and the sample input volume was 100 μL.

[0128] 2. According to the following steps, RNA was extracted using the chloroform-free RNA extraction kits provided in Examples 5 to 8 respectively:

[0129] 1) Take the sample cell suspension, centrifuge at room temperature and 1,000 rpm for 5 minutes, and discard the supernatant. Add 100 μL of enzyme-free water to resuspend the cell pellet to obtain the sample cell resuspension.

[0130] 2) Add 1 mL of chloroform-free RNA extraction lysis buffer to 100 μL of sample cell resuspension, vortex until fully dissolved, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 12,000 rpm for 15 minutes, and aspirate the supernatant (about 400 μL) into a new centrifuge tube to obtain the lysis product.

[0131] 3) Add an equal volume of isopropanol to the obtained supernatant (lysate), vortex to mix, and let stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature, carefully discard the supernatant, and retain the white RNA precipitate at the bottom of the tube.

[0132] 4) Add 1 mL of 75% ethanol aqueous solution and use a pipette to blow the precipitate several times to disperse it fully. Centrifuge at room temperature and 12,000 rpm for 10 minutes, carefully aspirate and discard the supernatant, and retain the white RNA precipitate at the bottom of the tube. Repeat step 4) once.

[0133] 5) Leave the obtained white RNA precipitate to dry at room temperature. Add 100 μL of enzyme-free water to dissolve the precipitate and incubate at 65°C for 10 min to fully dissolve the precipitate to obtain RNA solution.

[0134] The extracted RNA solution can be stored at -80°C for a long term, or at -20°C for a short term.

[0135] At the same time, the kit of Comparative Example 15 was selected and the sample RNA was extracted according to the operating method of the kit instruction manual.

[0136] 3. The extracted RNA solution was tested for purity using Nanodrop, concentration using Qubit, and integrity using Agilent4150. The specific experimental steps are as follows.

[0137] Nanodrop detection:

[0138] 1) Take the enzyme-free water used for elution in this experiment and perform Blank.

[0139] 2) After vortexing the obtained RNA solution, take 2 μL of RNA solution for measurement.

[0140] Qubit Assays:

[0141] 1) Prepare the required volume of Working Buffer by mixing Qubit RNA BR Reagent and Qubit RNA BR Buffer at a ratio of 1:199.

[0142] 2) Vortex the RNA solution to mix well, take 1 μL RNA solution into a Qubit assay tube, and add 199 μL Working Buffer into the same Qubit assay tube.

[0143] 3) Vortex and mix Qubit RNA BR Standard #1 and Qubit RNA BR Standard #2, respectively, and take 10 μL of the above solution into the Qubit assay tube, and add 190 μL of Working Buffer into the same Qubit assay tube.

[0144] 4) The mixtures in steps 2) and 3) were vortexed and centrifuged briefly, and incubated at room temperature for 2 minutes.

[0145] 5) After the incubation, the mixture in step 3) is placed in Qubit for standard curve calibration.

[0146] 6) After calibration, the mixture in step 2) is placed in Qubit for measurement.

[0147] Agilent 4150 Detection:

[0148] 1) Vortex the obtained RNA solution and transfer 1 μL of RNA solution to 8 tube strips.

[0149] 2) Vortex the RNA Ladder and transfer 1 μL of RNA Ladder to an 8-tube strip.

[0150] 3) Add 5 μL RNA Sample Buffer to the RNA Ladder and RNA solution.

[0151] 4) Place the 8-tube strip containing liquid in a vortex machine and oscillate at 2,000 rpm for 1 min.

[0152] 5) After shaking, centrifuge at 2,000 rpm for 1 min.

[0153] 6) After centrifugation, incubate the 8-tube strip at 72°C for 3 min, and then immediately place on ice for 2 min.

[0154] 7) After the ice bath, the treated solution was placed in Agilent 4150 for measurement.

[0155] The results are shown in Table 1 and Figure 1 shown.

[0156] Table 1

[0157]

[0158]

[0159] Among them, NanodropConc. is the sample concentration measured by ultraviolet absorption method, which means that Nanodrop estimates the nucleic acid concentration in the sample by measuring the absorbance at 260nm.

[0160] QubitConc. refers to the sample concentration measured using the Qubit Fluorometer from Thermo Fisher Scientific.

[0161] The A260 / A280 ratio is an indicator used to assess the degree of DNA, protein, phenol or guanidine contamination in the sample. A pure RNA sample should be close to 2.0. If this ratio is lower than the recommended value, it may be due to the presence of protein or other contaminants.

[0162] The A260 / A230 ratio is used to assess the degree of carbohydrate, phenol, or guanidine contamination in the sample. For pure nucleic acid samples, this ratio should generally be above 2.0. Lower ratios may indicate the presence of organic contamination.

[0163] From Table 1 and Figure 1 It can be seen that Examples 5 to 8 have an impact on the purity, concentration and RNA integrity (RIN e The values ​​of Example 1 and Example 5 are better than those of Example 15, among which Example 5 is the best.

[0164] Experimental Example 2

[0165] 1. Sample: 1×10 6 The K562 cell suspension was used as the experimental sample for RNA extraction, and the sample input volume was 100 μL.

[0166] 2. RNA was extracted using the chloroform-free RNA extraction kits provided in Example 5 and Comparative Examples 8 to 15, respectively. The specific operating steps were the same as those in Experimental Example 1.

[0167] The extracted RNA solution was tested for purity using Nanodrop, concentration using Qubit, and integrity using Agilent 4150. The subsequent experimental steps were the same as in Experimental Example 1, and the results are shown in Tables 2 and Figure 2 As shown:

[0168] Table 2

[0169]

[0170]

[0171] From Table 2 and Figure 2 It can be seen that Example 5 has an effect on the purity, concentration and RNA integrity (RIN e The quantitative comparison of values) are better than those of the comparative examples.

[0172] Compared with Example 5, the A260 / A280 of Comparative Example 8 is greater than 2.2, and the absence of a solvent and a separating agent reduces the stability of the lysate, affects the extraction and purification of the sample RNA, and increases the risk of DNA contamination or RNA degradation; Comparative Example 9 does not add a solvent, resulting in the inability to extract RNA normally; Comparative Example 10 does not add a separating agent, and its A260 / A280 is greater than 2.2, and A260 / A230 is less than 2, and the obtained RNA purity is low; the value of Nanodrop Conc. in Comparative Example 11 is close to twice the value of QubitConc., which shows that it contains high DNA contamination, and Comparative Example 12 cannot be extracted normally, which shows that even when a solvent and a separating agent are added, too high or too low a concentration of the chaotropic salt will affect the extraction of RNA; the Nanodrop Conc. values ​​of Comparative Examples 13 and 14 are close to those of Example 5, but the QubitConc. values ​​are lower than those of Example 5, which shows that too high or too low a reducing agent content may cause DNA contamination.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chloroform-free RNA extraction lysate, characterized in that: The invention comprises components with the following concentrations: 1-5 mol / L of chaotropic salt, 0.1%-5% (v / v) of anionic surfactant, 0.01-50 mmol / L of ion chelating agent, 0.01%-50% (v / v) of phenol solution, 30-300 mmol of buffer salt, 0.01%-1% (v / v) of reducing agent, 0.2%-1% (w / v) of auxiliary solvent, 10%-20% (w / v) of separating agent and 0.01%-0.2% (w / v) of fat-soluble indicator.

2. The chloroform-free RNA extraction lysate according to claim 1, characterized in that: The chaotropic salt comprises at least one of guanidine thiocyanate, ammonium thiocyanate, guanidine hydrochloride, sodium perchlorate, sodium periodate or sodium iodide; Preferably, the anionic surfactant includes at least one of sodium lauryl sarcosinate, sodium lauryl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate or dodecyl triethanolamine; Preferably, the ion chelating agent comprises at least one of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid or 8-hydroxyquinoline; Preferably, the phenol solution comprises at least one of water-saturated phenol, citric acid-saturated phenol or phenol; Preferably, the buffer salt comprises at least one of tris(hydroxymethylaminomethane), sodium citrate, sodium acetate or potassium acetate; Preferably, the reducing agent comprises at least one of β-mercaptoethanol, dithiothreitol, tris(2-carbonylethyl)phosphine hydrochloride or reduced glutathione; Preferably, the cosolvent comprises at least one of dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran; Preferably, the separating agent comprises at least one of hexamethylphosphoramide or tetramethylethylenediamine; Preferably, the fat-soluble indicator comprises at least one of solvent blue 36, solvent blue 38, bromocresol green or phenol red.

3. The chloroform-free RNA extraction lysate according to claim 1 or 2, characterized in that: The pH of the chloroform-free RNA extraction lysis solution is 3-5.

4. The method for preparing a chloroform-free RNA lysate according to any one of claims 1 to 3, characterized in that: The preparation method is prepared by dissolving each component in water according to the formula amount.

5. Use of the chloroform-free RNA extraction lysate according to any one of claims 1 to 3 or the chloroform-free RNA extraction lysate prepared by the preparation method according to claim 4 in RNA extraction.

6. A kit for extracting RNA without chloroform, characterized in that: The invention comprises the chloroform-free RNA extraction lysate according to any one of claims 1 to 3 or the chloroform-free RNA extraction lysate prepared by the preparation method according to claim 4.

7. The kit according to claim 6, characterized in that The kit also includes RNA extraction solution; Preferably, the RNA extraction solution comprises isopropanol; Preferably, the kit further comprises an RNA elution solution; Preferably, the RNA elution solution comprises an alcohol solution or water.

8. A method for extracting RNA without chloroform, characterized in that: The method comprises the following steps: A. Add the chloroform-free RNA extraction lysis solution according to any one of claims 1 to 3 or the chloroform-free RNA extraction lysis solution prepared by the preparation method according to claim 4 to the sample cell suspension, shake and dissolve, then stand, and centrifuge to obtain a lysis product; B. Mix the RNA extract and the lysate, incubate, and centrifuge to obtain RNA precipitate.

9. The method according to claim 8, characterized in that The method for preparing the sample cell suspension comprises taking a cell suspension, centrifuging to obtain a precipitate, and adding water to resuspend the precipitate to obtain a sample cell suspension; Preferably, the cell amount of the sample cell suspension is less than 5×10 7 ; Preferably, the amount of the chloroform-free RNA extraction lysis solution used is 1-2 mL of chloroform-free RNA extraction lysis solution per 100 μL of sample cell suspension, preferably 1 mL; Preferably, the RNA extract and the lysate are mixed in equal volumes; Preferably, the standing time in step A is 3 to 10 minutes; Preferably, the centrifugal speed in step A is 10000-14000 rpm, preferably 12000 rpm; Preferably, the centrifugation time in step A is 12 to 18 minutes, preferably 15 minutes; Preferably, the centrifugation temperature in step A is 0-6°C, preferably 4°C; Preferably, the incubation temperature in step B is 25-37°C; Preferably, the incubation time in step B is 3 to 10 minutes; Preferably, the centrifugal speed in step B is 10000-14000 rpm, preferably 12000 rpm; Preferably, the centrifugation time in step B is 8 to 15 min, preferably 10 min.

10. The method according to claim 8 or 9, characterized in that: The method further comprises step C, dispersing the RNA precipitate in an RNA elution solution and centrifuging to obtain the RNA precipitate; Preferably, the amount of the RNA elution solution is 1-2 mL of RNA elution solution per 100 μL of sample cell suspension, preferably 1 mL; Preferably, the operation of step C is repeated 1 to 2 times; Preferably, after step C, the method further comprises dissolving the dried RNA precipitate in water and incubating to obtain an RNA solution; Preferably, the water used to dissolve the RNA precipitate is added in equal volumes as the sample cell suspension; Preferably, the incubation temperature in step C is 60-70°C, preferably 65°C; Preferably, the incubation time in step C is 2 to 5 minutes.

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