Plant RNA extraction reagents and applications

Abstract

The present invention relates to the field of plants, and in particular to plant RNA extraction reagents and applications. The present invention provides a sample processing solution, comprising: component 1 and component 2; the component 1 comprises: a grinding agent; the component 2 comprises: a lysate. The present invention configures a set of reagents that can effectively remove the influence of polysaccharides and polyphenols to obtain RNA with high purity and good integrity. The reagent of the present invention does not contain any toxic or irritating odor substances. The reagent of the present invention can realize high-throughput automated extraction. In the field of plant physiological detection and research, the reagent can be used to extract various types of plants to achieve the purpose of effectively separating and purifying RNA.

Description

Technical Field

[0001] The present invention relates to the field of plants, and in particular to a plant RNA extraction reagent and application thereof. Background Art

[0002] Plant cells contain cell walls, the main components of which are polysaccharides such as cellulose and pectin. The cell walls have high mechanical strength and strict requirements for lysis. Some plant cells contain a large number of secondary metabolites, mainly phenolic substances. During the extraction and purification process, phenols are easily oxidized to quinones, which irreversibly bind to RNA, affecting the final purity and subsequent experiments.

[0003] Common plant RNA extraction methods:

[0004] 1. Trizol method: This method uses the strong deformation and oxidation ability of guanidine isothiocyanate or phenol in the extract to lyse the cell membrane, denature the protein, inhibit the activity of endogenous RNase, and release the nucleic acid into the aqueous phase. RNA is then recovered by precipitation. However, when this method is used to extract plants with high polyphenol content, phenolic substances will be oxidized into brown quinone compounds, which will irreversibly bind to nucleic acids. Using chloroform to remove polyphenols will cause serious loss of RNA. In addition, reagents such as phenol and chloroform are toxic to a certain extent, which endangers the safety of experimenters.

[0005] 2. SDS-phenol method: SDS and phenol are used to lyse cell membranes, denature proteins, inhibit endogenous RNase activity, and precipitate RNA as white flocculent precipitates to remove DNA and proteins. This method will precipitate a small amount of DNA when extracting nucleic acids, resulting in residual DNA in the subsequent extraction products, affecting subsequent downstream experiments. It is also unable to effectively separate phenolic impurities, resulting in low purity of the extraction product.

[0006] 3. Kit method: There are many kits for plant RNA extraction on the market. The lysis solution in the kit lyses cells, guanidine hydrochloride inhibits RNase activity, and β-mercaptoethanol prevents phenolic substances from being oxidized. Finally, the adsorption membrane on the centrifugal column specifically adsorbs RNA to complete the purification. Currently, most of the extraction methods on the market are silica gel column extraction methods, which cannot be automated. In addition, irritating odor substances such as DTT and β-mercaptoethanol are used. This type of strong reducing agent has poor stability and needs to be refrigerated.

[0007] Therefore, there is a need for a reagent and method that can effectively remove the effects of polysaccharides and polyphenols and produce RNA with high purity and good integrity. Summary of the invention

[0008] In view of this, the present invention provides a plant RNA extraction reagent and application. The present invention is configured with a set of reagents that can effectively remove the influence of polysaccharides and polyphenols to obtain RNA with high purity and good integrity. The reagent of the present invention does not contain any toxic or irritating odor substances. The reagent of the present invention can achieve high-throughput automated extraction. In the field of plant physiological detection and research, the reagent can be used to extract various types of plants to achieve the purpose of effectively separating and purifying RNA.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a sample processing solution, comprising: component 1 and component 2;

[0011] The component 1 comprises: an abrasive, wherein the abrasive comprises: 60% (m / m) cross-linked polyvinyl pyrrolidone and 40% (m / m) polyethylene glycol;

[0012] The component 2 includes: a lysate, wherein the lysate includes the following components:

[0013]

[0014] In some embodiments of the present invention, in the sample processing solution, the reducing agent includes: sodium citrate or DTT.

[0015] In some embodiments of the present invention, in the sample processing solution, the reducing agent is sodium citrate.

[0016] The present invention also provides an extraction reagent, comprising: components 3 to 8;

[0017] The component 3 comprises: 20-100 mg / mL magnetic beads;

[0018] The component 4 comprises: a proteinase K solution;

[0019] The component 5 includes: a binding liquid, wherein the binding liquid includes anhydrous ethanol;

[0020] The component 6 comprises: an endonuclease;

[0021] The component 7 includes an eluent, and the eluent includes: 1mM to 50mM tris(hydroxymethyl)aminomethane;

[0022] The component 8 comprises a washing liquid, and the washing liquid comprises the following components:

[0023]

[0024]

[0025] In some embodiments of the present invention, in the above-mentioned extraction reagent, the magnetic beads include: super-cis-silica hydroxyl magnetic beads or super-cis-silica carboxyl magnetic beads.

[0026] In some embodiments of the present invention, in the above-mentioned extraction reagent, the magnetic beads are: super-cis-silica hydroxy magnetic beads.

[0027] In some embodiments of the present invention, in the above-mentioned extraction reagent, the polyvinyl alcohol PEG includes: PEG-4000, PEG-2000 or PEG-8000.

[0028] In some embodiments of the present invention, in the above-mentioned extraction reagent, the polyvinyl alcohol PEG is: PEG-4000.

[0029] The present invention also provides a plant RNA extraction reagent, comprising: the sample processing solution and the extraction reagent.

[0030] In some embodiments of the present invention, the above-mentioned plant RNA extraction reagent further includes: a buffer solution.

[0031] In some embodiments of the present invention, in the above-mentioned plant RNA extraction reagent, the buffer comprises: 10×Reaction Buffer.

[0032] The present invention also provides a plant RNA extraction kit, comprising: the above-mentioned sample processing solution, the above-mentioned extraction reagent and / or the above-mentioned plant RNA extraction reagent and an acceptable auxiliary agent and / or a carrier.

[0033] The present invention also provides the use of the sample processing solution, the extraction reagent, the plant RNA extraction reagent and / or the plant RNA extraction kit in extracting plant RNA.

[0034] The present invention also provides a method for extracting plant RNA, which comprises mixing the plant RNA extraction reagent and / or the plant RNA extraction kit with a sample.

[0035] In some embodiments of the present invention, in the above extraction method, the mixing comprises the following steps:

[0036] S1: Sample processing: the sample is mixed with the component 1, ground, mixed with the component 2, centrifuged, and the supernatant is collected;

[0037] S2: Combine: Mix the supernatant collected in S1 with component 3, component 4 and component 5, separate and collect the supernatant;

[0038] S3: washing: mixing the supernatant collected in S2 with the component 8, and collecting the supernatant;

[0039] S4: digestion: the supernatant collected in S3 is mixed with the component 6, and the precipitate is collected;

[0040] S5: Elution: The precipitate collected in S4 is dried and mixed with the component 7 to obtain the RNA of the sample.

[0041] In some embodiments of the present invention, in the above extraction method, the mass ratio of the sample to the component 1 is 1:10.

[0042] In some embodiments of the present invention, the separation time in the above extraction method S2 is no less than 2 minutes.

[0043] The plant RNA extraction kit provided by the present invention does not contain any toxic or irritating odor substances, thereby protecting the safety of experimenters. At the same time, cross-linked polyvinyl pyrrolidone and polyethylene glycol are used as grinding agents to remove polyphenols during grinding, and high-concentration potassium acetate is used to precipitate and remove polysaccharides during centrifugation, thereby eliminating the influence of polysaccharides and polyphenols on extraction, thereby ensuring that the magnetic beads are completely combined with RNA, and obtaining RNA with high purity and integrity. The present invention can also realize automated extraction, and realize high-throughput and stable extraction of RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0045] Figure 1 The agarose gel electrophoresis diagram after extracting plant RNA in Example 2 is shown;

[0046] Figure 2 The agarose gel electrophoresis diagram after extracting plant RNA in Example 3 is shown;

[0047] Figure 3 The agarose gel electrophoresis diagram after extracting plant RNA in Comparative Example 1 is shown;

[0048] Figure 4 The agarose gel electrophoresis diagram after extracting plant RNA in Comparative Example 2 is shown;

[0049] Figure 5 The agarose gel electrophoresis diagram after extracting plant RNA in Example 4 is shown;

[0050] Figure 6 The agarose gel electrophoresis diagram after plant RNA extraction in Comparative Example 3 is shown. DETAILED DESCRIPTION

[0051] The invention discloses a plant RNA extraction reagent and application.

[0052] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0053] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0054] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0055] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0056] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0057] The invention provides a kit and method for extracting plant RNA which can precipitate polysaccharides, remove polyphenols, and obtain high-purity and high-concentration plant RNA.

[0058] The plant RNA extraction reagent provided by the present invention comprises: a grinding agent, magnetic beads, DNase I, 10×Reaction Buffer, proteinase K, lysate, washing solution and elution solution;

[0059] The abrasive includes: cross-linked polyvinyl pyrrolidone (1% to 10% of the sample mass) and polyethylene glycol (1% to 10% of the sample mass).

[0060] The lysate comprises: KAc (0.1-5M), ethylenediaminetetraacetic acid (0.1-0.5mM), GuHCl (1-5M), sodium citrate (20-200mM), Tween-20 (0.1%-10% (V / V)), CTAB (1%-5% (m / V)) and DEPC water;

[0061] The eluent includes: tris(hydroxymethyl)aminomethane hydrochloride and DEPC water

[0062] The washing solution comprises: GuSCN, GuHCl, PEG-4000, ethylenediaminetetraacetic acid, KAc, Tween-20, propanol and DEPC water;

[0063] The reagent provided by the present invention does not need to use toxic reagents such as DTT and β-mercaptoethanol, thereby avoiding harm to the human body. The grinding agent can be added during grinding to remove plant polyphenols, the high concentration of KAc in the lysate can remove plant polysaccharides in the centrifugation step, and the components such as guanidine salt and proteinase K in the lysate can lyse plant cells under normal temperature conditions to avoid affecting plant RNA; different concentrations of guanidine salt are used during washing to effectively remove plant proteins, salts and other small molecule compounds, thereby obtaining high-quality and complete plant RNA.

[0064] The detailed steps are:

[0065] A. Operation steps

[0066] 1. Preparation:

[0067] Anhydrous ethanol

[0068] EP tube (1.5mL centrifuge tube, 3 per sample)

[0069] Single channel pipette: 20μL, 200μL, 1000μL

[0070] Vortex oscillator, constant temperature metal bath (or water bath)

[0071] Magnetic separator: Beaver magnetic separator can be used, item number: 60201

[0072] 2. Operation steps

[0073] 1) Prepare DNase I reaction solution

[0074] Take a 1.5mL centrifuge tube and add 80×NμL of elution buffer, 10×NμL of 10×Reaction Buffer, and 10×NμL of DNase I in sequence according to the number of samples N. Use a pipette to slowly pipette 20 times or slowly invert the centrifuge tube 20 times to mix well and set aside.

[0075] 2) Grinding of plant samples (taking 50 mg sample as an example)

[0076] Take a sufficient amount of fresh plant samples or frozen samples, add grinding agent with a ratio of 1 / 10 of the plant mass, and then add liquid nitrogen to grind them thoroughly.

[0077] 3) Lysis

[0078] Add 50-100 mg of ground plant sample to a 1.5 mL centrifuge tube, add 600 μL of lysis buffer, adjust the speed to vortex for 1 min, mix thoroughly and let stand at room temperature for 5 min. Centrifuge at 13000 rpm for 10 min, transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube.

[0079] 4) Combination

[0080] Add 160 μL of anhydrous ethanol, 20 μL of magnetic beads, and 20 μL of proteinase K to the above centrifuge tube in sequence, shake and mix for 30 seconds, bind at room temperature for 5 minutes, invert and mix twice during the process, then place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0081] Note: The magnetic separation time in this step should be no less than 2 minutes.

[0082] 5) Washing

[0083] a. Add 600 μL of washing solution to a 1.5 mL centrifuge tube, shake and mix for 30 seconds, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0084] b. Add 600 μL of 80% ethanol to each centrifuge tube, vortex and shake at the maximum speed of the vortex oscillator for 2 minutes, place the centrifuge tube on a magnetic separator until the solution is clear, discard the supernatant with a pipette, let it stand at room temperature for 3 minutes, and remove the centrifuge tube.

[0085] 6) DNA digestion

[0086] a. Add 100 μL of DNase I reaction solution into each centrifuge tube. Use a pipette to draw up the DNase I reaction solution in the tube and repeatedly blow the magnetic beads on the tube wall. After confirming that all the magnetic beads on the tube wall have been blown into the reaction solution, slowly pipette the reaction solution 20 times to resuspend the magnetic beads.

[0087] b. Leave at room temperature for 15 to 30 minutes.

[0088] 7) Washing

[0089] Remove the 1.5 mL centrifuge tube from the magnetic stand, add 600 μL 80% ethanol, vortex for 30 seconds, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube. Repeat this operation once more.

[0090] 8) Drying

[0091] Keep the centrifuge tube on the magnetic separator and air-dry it at room temperature until the surface of the magnetic beads has no obvious gloss (10 minutes), then remove the centrifuge tube.

[0092] Note: If any liquid is found to remain in the reaction tube during the drying process, use a small-range pipette to discard the liquid.

[0093] 9) Elution

[0094] Add 50-100 μL of elution buffer, shake and mix for 30 seconds, let stand at room temperature for 5 minutes, place the centrifuge tube on a magnetic separator until the solution becomes clear, and transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified plant RNA, which can be stored at -20°C.

[0095] B. Automated operation steps

[0096] Preparation:

[0097] 1 magnetic rod set, 1 96-well deep well plate

[0098] Nucleic acid extraction equipment: BeaverRosetta32 automated nucleic acid extraction instrument

[0099] 1.5mL centrifuge tube (DNase-free, RNase-free)

[0100] Single channel pipette: 20μL, 200μL, 1000μL, 5000μL

[0101] Vortex mixer

[0102] Anhydrous ethanol (analytical grade)

[0103] Note: Before each use, the kit must be equilibrated to room temperature.

[0104] 1. Confirm that the instrument status and program are normal (users can adjust the extraction program parameters according to actual usage, such as magnetic adsorption time, drying time, elution volume, etc.).

[0105] 2. Add 50-100 mg of ground plant sample to a 1.5 mL centrifuge tube, add 600 μL of lysis buffer, adjust the speed to a suitable vortex oscillation for 1 min, mix thoroughly and let stand at room temperature for 5 min. Centrifuge at 13000 rpm for 10 min.

[0106] 3. Add reagents and samples

[0107] Follow these steps:

[0108] a.1,7 columns, add 160 μL of anhydrous ethanol, 20 μL of magnetic beads, 20 μL of proteinase K, and 400 μL of the supernatant after centrifugation in step 2 to each well.

[0109] b.2, 8 columns, add 600 μL of washing solution to each well.

[0110] c.3,9 columns, add 100 μL DNase I reaction solution to each well.

[0111] d. Add 600 μL of 80% anhydrous ethanol to each well in columns 4, 5, 10, and 11.

[0112] e.6,12 columns, add 100 μL of elution buffer to each well.

[0113] f. Place the deep-well plate into the extractor, make sure the deep-well plate is completely inserted into the slot, and insert the magnetic rod sleeve.

[0114] 4. Immediately run the preset program in the instrument.

[0115] 5. After the program is finished, immediately transfer the eluate in columns 6 and 12 to the corresponding 1.5mL centrifuge tubes. This is the purified nucleic acid.

[0116] The lysate in step 2) comprises the following components:

[0117] 0.5-5 M guanidine thiocyanate;

[0118] 0.1~3M potassium acetate;

[0119] 0.1~10% (V / V) Tween-20;

[0120] 1-20 mM disodium EDTA;

[0121] 1%~5%(g / L)CTAB;

[0122] 0.1~5% (g / L) sodium citrate;

[0123] DEPC water.

[0124] In step 3), the superparamagnetic silica beads are hydroxy magnetic beads with a concentration of 20 to 100 mg / mL.

[0125] In step 3), the washing liquid comprises the following components:

[0126] 0.5-4 M guanidine thiocyanate;

[0127] 0.1-2M guanidine hydrochloride;

[0128] 1-10% (g / L) polyvinyl alcohol PEG 4000;

[0129] 0.01~0.5% (V / V) Tween-20;

[0130] 1~50mM sodium acetate;

[0131] 1~50mM disodium EDTA;

[0132] 10-60% (V / V) isopropyl alcohol;

[0133] DEPC water.

[0134] In step 3), the eluent comprises the following components:

[0135] 1~50mM tris(hydroxymethyl)aminomethane;

[0136] DEPC water.

[0137] In Examples 1 to 4 of the present invention and Comparative Examples 1 to 3, all the raw materials and reagents used can be purchased from the market.

[0138] The present invention will be further described below in conjunction with embodiments:

[0139] Example 1

[0140] Prepare a plant RNA extraction kit, including grinding agent, superparamagnetic silica hydroxy magnetic beads, DNaseⅠ, 10× Reaction Buffer, proteinase K solution, lysis solution, binding solution, washing solution and elution solution.

[0141] The superparamagnetic silica hydroxy magnetic beads have a diameter of 200 nm and a concentration of 20 mg / mL.

[0142] The concentrations of DNase I and proteinase K were both 10 mg / mL.

[0143] Abrasives include: 60% cross-linked polyvinyl pyrrolidone and 40% polyethylene glycol

[0144] The lysis buffer includes: 200 mM KAc (pH 5.0), 35 mM EDTA, 5 M GuHcl, 40 mM sodium citrate, 2% CTAB, and 10% Tween-20.

[0145] The binding solution includes: anhydrous ethanol.

[0146] The washing solution includes 3M guanidine thiocyanate, 5% PEG4000, 0.02% Tween-20, 20mM sodium acetate, 25mM disodium ethylenediaminetetraacetate, and 45% isopropanol.

[0147] The eluent was 10 mM Tris(OH)2.

[0148] Example 2

[0149] The kit of Example 1 is used to extract plant samples, and the extraction scheme includes the following steps:

[0150] 1. Sample preparation: Take 2 clean rice samples and 2 rapeseed samples, 50 mg each, add the samples and 5 mg grinding agent into a clean mortar, add liquid nitrogen and grind into powder with a pestle, and transfer the powder completely to a 1.5 mL centrifuge tube.

[0151] (1) Add 600 μL of lysis buffer to the sample, vortex for 1 min, mix thoroughly, let stand at room temperature for 5 min, centrifuge at 13,000 rpm for 10 min, and transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube.

[0152] (2) Add 160 μL of anhydrous ethanol, 20 μL of magnetic beads, and 20 μL of proteinase K to the above centrifuge tube in sequence, shake and mix for 30 seconds, allow to bind at room temperature for 5 minutes, invert and mix twice, then place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0153] (3) Add 600 μL of washing solution to a 1.5 mL centrifuge tube and vortex to mix for 30 seconds. Place the centrifuge tube on a magnetic separator until the solution becomes clear. Remove the supernatant with a pipette and remove the centrifuge tube.

[0154] (4) Add 600 μL of 80% ethanol to each centrifuge tube and vortex for 2 min at the maximum speed of a vortex oscillator. Place the centrifuge tube on a magnetic separator until the solution becomes clear. Discard the supernatant with a pipette and let it stand at room temperature for 3 min. Remove the centrifuge tube.

[0155] (5) Add 100 μL of DNase I reaction solution to each centrifuge tube. Use a pipette to draw up the DNase I reaction solution in the tube and repeatedly blow the magnetic beads on the tube wall. After confirming that all the magnetic beads on the tube wall have been blown into the reaction solution, slowly pipette the reaction solution 20 times to resuspend the magnetic beads.

[0156] (6) Remove the 1.5 mL centrifuge tube from the magnetic stand, add 600 μL of 80% ethanol, vortex for 30 seconds, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube. Repeat this operation once more.

[0157] (7) Keep the centrifuge tube on the magnetic separator and air-dry it at room temperature until the surface of the magnetic beads has no obvious gloss (10 min).

[0158] (8) Add 50-100 μL of elution buffer and vortex to mix for 30 seconds. After standing at room temperature for 5 minutes, place the centrifuge tube on a magnetic separator until the solution becomes clear. Transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified plant RNA, which can be stored at -20°C.

[0159] 2. RNA content and purity determination in the eluate and agarose gel electrophoresis results are shown in Tables 1 and Figure 1 shown.

[0160] Table 1 Determination of RNA content and purity in the eluate

[0161]

[0162]

[0163] Experimental results: RNA with high purity, high content (>20 ng / μL) and bright electrophoresis bands can be extracted using the kit of the present invention.

[0164] Example 3

[0165] The kit of Example 1 was used as Experiment A, and three sets of reagents were added.

[0166] Group B: Lysis buffer included: 50 mM KAc (pH 5.0), 35 mM EDTA, 5 M GuHcl, 40 mM sodium citrate, 2% CTAB, and 10% Tween-20.

[0167] Group C: Lysis buffer included: 5M KAc (pH 5.0), 35mM EDTA, 5M GuHcl, 40mM sodium citrate, 2% CTAB, and 10% Tween-20.

[0168] Group D: Lysis buffer included: 35 mM EDTA, 5 M GuHcl, 40 mM sodium citrate, 2% CTAB, and 10% Tween-20.

[0169] The remaining components were consistent with group A.

[0170] The extraction scheme adopts the method in Example 2 to extract the watermelon pulp.

[0171] 2. RNA content and purity determination in the eluate and agarose gel electrophoresis results are shown in Table 2 and Figure 2 shown.

[0172] Table 2 Determination of RNA content and purity in the eluate

[0173]

[0174] Experimental results: The extraction effect of potassium acetate was excellent within the concentration range, with group A being the best. The extraction effect of group D without the addition of potassium acetate was poor and failed to effectively remove polysaccharides.

[0175] Comparative Example 1

[0176] No grinding agent was added in the sample grinding step, the sample type was replaced by grape skin, and the other reagents and steps were the same as in Example 1.

[0177] The concentration, purity and fragment size of the extracted products obtained in the examples and comparative examples were tested according to the above method. The results are shown in Table 3 and Figure 3 shown.

[0178] Table 3 Determination of RNA content and purity in the eluate

[0179]

[0180] Experimental results: Compared with the control example without adding abrasive, the use of the kit of Example 1 can effectively remove the influence of polyphenols on extraction and extract RNA with higher purity.

[0181] Comparative Example 2

[0182] The effects of different concentrations of potassium acetate lysis solutions on the extraction of RNA from polysaccharide plants were investigated: the sample was replaced with watermelon pulp, and the lysis solution used a low concentration of 50 mM KAc instead of the original 200 mM KAc, and the remaining steps and solutions were the same as in Examples 1-2.

[0183] The concentration and purity of the extracted products obtained in Example 4 and Comparative Example 2 were tested according to the above method. The results are shown in Table 4 and Figure 4 shown.

[0184] Table 4 Determination of RNA content and purity in the eluate

[0185]

[0186] Experimental results: The potassium acetate lysis solution of the present invention can effectively remove polysaccharide impurities and obtain relatively pure DNA.

[0187] Example 4

[0188] The kit of Example 1 is used to automatically extract plant samples, and the extraction scheme includes the following steps:

[0189] 1. Sample preparation: Take 4 clean rice leaf samples, 50 mg each, add the sample and 5 mg grinding agent in a clean mortar, add liquid nitrogen and grind into powder with a pestle, and transfer the powder completely to a 1.5 mL centrifuge tube.

[0190] (1) Take a 96-well deep-well plate and dispense the corresponding reagents according to the following specifications

[0191] a.1,7 columns, add 160μL anhydrous ethanol, 20μL magnetic beads, and 20μL proteinase K to each well.

[0192] b.2, 8 columns, add 600 μL of washing solution to each well.

[0193] c.3,9 columns, add 100 μL DNase I reaction solution to each well.

[0194] d. Add 600 μL of 80% anhydrous ethanol to each well in columns 4, 5, 10, and 11.

[0195] e.6,12 columns, add 100 μL of elution buffer to each well.

[0196] (2) Add 600 μL of lysis buffer to the sample, vortex for 1 min, mix thoroughly, let stand at room temperature for 5 min, centrifuge at 13,000 rpm for 10 min, and transfer 400 μL of supernatant to columns 1 and 7.

[0197] (3) Place the deep-well plate into the extractor, make sure that the deep-well plate is completely inserted into the slot, insert the magnetic rod sleeve, and run the program.

[0198] (4) After the program is finished, immediately transfer the eluate in columns 6 and 12 to the corresponding 1.5 mL centrifuge tubes. This is the purified nucleic acid.

[0199] 2. RNA content and purity determination in the eluate and agarose gel electrophoresis results are shown in Table 5 and Figure 5 shown.

[0200] Table 5 Determination of RNA content and purity in the eluate

[0201] serial number Concentration ng / μL Extraction amount μg 260 / 280 260 / 230 1 100 10.00 2.08 2.05 2 100.04 10.00 2.09 2.04 3 102.12 10.21 2.09 2.03 4 101.36 10.14 2.11 2.03

[0202] Experimental results: The automated extraction method of the present invention can stably and quickly extract relatively pure plant RNA.

[0203] Comparative Example 3

[0204] The effects of different binding conditions on plant RNA extraction were investigated: the sample was rice leaves, and the binding solution was set to 40% ethanol as group C;

[0205] Binding solution A: 3M guanidine thiocyanate + 5% PEG + 1M NaCl + 0.2M NaAc

[0206] Binding solution B: 3M guanidine hydrochloride + 5% PEG + 1M NaCl + 0.2M NaAc

[0207] The remaining steps and solutions are the same as those in Examples 1-2.

[0208] The concentration and purity of the extracted products obtained in Example 6 and Comparative Example 3 were tested according to the above method. The results are shown in Table 6 and Figure 6 shown.

[0209] Table 6 Determination of RNA content and purity in the eluate

[0210] Group Concentration ng / μL 260 / 280 260 / 230 Group A 100.04 2.11 2.22 Group B 99.08 2.14 2.21 Group C 126.51 2.12 2.20

[0211] Experimental results: The presence of different salts in the binding solution had little effect on the final concentration, but replacing the binding solution with ethanol could effectively increase the extraction concentration.

[0212] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sample processing solution, characterized in that: include: Component 1 and component 2; The component 1 comprises: an abrasive, wherein the abrasive comprises: 60% (m / m) cross-linked polyvinyl pyrrolidone and 40% (m / m) polyethylene glycol; The component 2 includes: a lysate, wherein the lysate includes the following components:

2. Extraction reagent, characterized in that include: Component 3 to Component 8; The component 3 comprises: 20-100 mg / mL magnetic beads; The component 4 comprises: a proteinase K solution; The component 5 includes: a binding liquid, wherein the binding liquid includes anhydrous ethanol; The component 6 comprises: an endonuclease; The component 7 includes: an eluent, wherein the eluent includes: 1 mM to 50 mM tris(hydroxymethyl)aminomethane; The component 8 includes: a washing liquid, and the washing liquid includes the following components:

3. A plant RNA extraction reagent, characterized in that: include: The sample processing solution as claimed in claim 1 and the extraction reagent as claimed in claim 2.

4. The plant RNA extraction reagent according to claim 3, characterized in that Also includes: Buffer.

5. A plant RNA extraction kit, characterized in that: include: The sample processing solution according to claim 1, the extraction reagent according to claim 2 and / or the plant RNA extraction reagent according to claim 3 or 4, and an acceptable auxiliary agent and / or carrier.

6. Use of the sample processing solution according to claim 1, the extraction reagent according to claim 2, the plant RNA extraction reagent according to claim 3 or 4 and / or the plant RNA extraction kit according to claim 5 in extracting plant RNA.

7. A method for extracting plant RNA, characterized in that: The plant RNA extraction reagent according to claim 3 or 4 and / or the plant RNA extraction kit according to claim 5 is mixed with the sample.

8. The extraction method according to claim 7, characterized in that The mixing comprises the following steps: S1: Sample processing: the sample is mixed with the component 1, ground, mixed with the component 2, centrifuged, and the supernatant is collected; S2: Combine: Mix the supernatant collected in S1 with component 3, component 4 and component 5, separate and collect the supernatant; S3: washing: mixing the supernatant collected in S2 with the component 8, and collecting the supernatant; S4: digestion: the supernatant collected in S3 is mixed with the component 6, and the precipitate is collected; S5: Elution: The precipitate collected in S4 is dried and mixed with the component 7 to obtain the RNA of the sample.

9. The extraction method according to claim 8, characterized in that The mass ratio of the sample to the component 1 is 10:

1.

10. The extraction method according to claim 8 or 9, characterized in that The separation time described in S2 is not less than 2 minutes.

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