A method for rapidly extracting total RNA from plant tissues based on magnetic bead method

Abstract

This application relates to the field of RNA extraction technology, specifically to a method for rapid extraction of total RNA from plant tissues based on magnetic beads. This method is applicable to the extraction of total RNA from plant tissues such as roots, stems, leaves, flowers, and fruits. It allows for the acquisition of high-quality total RNA samples through a relatively simple and rapid experimental process, making it suitable for quantitative PCR and transcriptome sequencing, and thus possessing significant application value.

Description

Technical Field

[0001] This application relates to the field of RNA extraction technology, specifically to a method for rapidly extracting total RNA from plant tissues based on magnetic beads. Background Technology

[0002] RNA extraction from plant tissues is a necessary prerequisite for plant molecular biology research. High-quality RNA is required for molecular biology studies such as Northern blotting, mRNA purification for in vitro translation or cDNA library construction, RT-PCR, and differential analysis. Therefore, extracting high-purity, intact RNA from plant tissues is crucial for the successful conduct of these studies.

[0003] Most plant tissues are rich in phenolic compounds, polysaccharides, and certain unidentified secondary metabolites, as well as exhibiting high RNase activity, making it difficult to effectively isolate and purify RNA from plant tissues. When plant tissues are ground and cells are broken down, these substances interact with RNA. Phenolic compounds, after being oxidized to quinones, irreversibly bind to RNA; polysaccharides form insoluble colloids that co-precipitate with RNA; terpenoids and RNases cause chemical degradation and enzymatic hydrolysis of RNA, respectively, resulting in low RNA yield and poor quality. For these types of plant tissue samples, conventional RNA extraction methods (such as the guanidine method, phenol method, and hexadecyltrimethylammonium bromide method) are insufficient to extract RNA. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this application is to provide a method for rapid extraction of total RNA from plant tissues based on magnetic beads, which solves the problems of cumbersome steps, numerous reagents, and low extraction rates in the existing technology for extracting total RNA from plant tissues. The method of this application is applicable to the extraction of total RNA from plant tissues such as roots, stems, leaves, flowers, and fruits. It can obtain high-quality total RNA samples through a relatively simple and rapid experimental process, making it suitable for quantitative PCR and transcriptome sequencing, and has significant application value.

[0005] To achieve the above and other related objectives, the first aspect of this application provides a kit for extracting total RNA from plant tissues, comprising one or more of the following raw materials:

[0006] Lysis buffer: comprising one or more combinations of 2-6M guanidine isothiocyanate, 0.01-0.3M boric acid, 50-500mM tris(hydroxymethyl)aminomethane-hydrochloric acid, 20-50mM sodium chloride, and 20-50mM disodium ethylenediaminetetraacetate;

[0007] Potassium acetate solution: The solutes include 2.5M potassium acetate and / or 0.5M potassium chloride;

[0008] Magnetic bead solution: comprising magnetic beads, wherein the magnetic beads are selected from silanol magnetic beads or carboxyl magnetic beads;

[0009] Protein removal buffer: Solutes include protein denaturants and / or salt ions;

[0010] Nucleic acid elution buffer: The solute includes 0.1% diethyl pyrocarbonate.

[0011] In any embodiment of this application, the kit further includes one or more of the following: rinsing buffer, phenol, chloroform, and anhydrous ethanol; preferably, the rinsing buffer is an ethanol solution.

[0012] In any embodiment of this application, the pH of the tris(hydroxymethyl)aminomethane-hydrochloric acid in the lysis buffer is 7.0-7.5.

[0013] In any embodiment of this application, the pH of potassium chloride in the potassium acetate solution is 4.8-5.2.

[0014] In any embodiment of this application, the protein denaturing agent in the protein removal buffer includes one or more combinations of urea, guanidine salts, and proteinase K; preferably, the guanidine salts include guanidine isothiocyanate and / or guanidine hydrochloride.

[0015] In any embodiment of this application, the salt ions in the protein removal buffer include one or more combinations of LiCl, NaCl, and KCl.

[0016] In any embodiment of this application, the volume ratio of phenol to chloroform is 3:2.

[0017] In any embodiment of this application, the solute of the protein removal buffer includes 2-4M guanidine isothiocyanate and / or 0.5-2M LiCl.

[0018] The second aspect of this application provides a method for extracting total RNA from plant tissues, using the kit described in the first aspect, comprising the following steps:

[0019] 1) Grind the plant tissue into powder, add lysis buffer and potassium acetate solution to obtain the first treatment product;

[0020] 2) Centrifuge the first treated product, take the supernatant, add phenol and chloroform to obtain the second treated product;

[0021] 3) Centrifuge the second-processed material, take the supernatant, add anhydrous ethanol and magnetic bead solution, mix well, and then separate the magnetic beads;

[0022] 4) After mixing the magnetic beads with the protein removal buffer, separate the magnetic beads;

[0023] 5) After mixing the magnetic beads with the rinsing buffer, separate the magnetic beads;

[0024] 6) Elute the magnetic beads with nucleic acid elution buffer to obtain total RNA from plant tissue.

[0025] In any embodiment of this application, in step 1), the plant tissue is selected from the root, stem, leaf, flower, or fruit tissue of a plant.

[0026] In any embodiment of this application, in step 1), the grinding is performed in liquid nitrogen; the grinding is performed in a grinding machine with a frequency of 45-55 Hz and a grinding time of 15-20 s.

[0027] In any embodiment of this application, in step 1), after adding lysis buffer and potassium acetate solution, the mixture is allowed to stand to obtain the first processed product; preferably, the first standing time is 2-3 minutes.

[0028] In any embodiment of this application, in step 2), the centrifugation conditions are 4°C, 15000g, and centrifugation time is 2-5min.

[0029] In any embodiment of this application, in step 2), phenol and chloroform are added and then allowed to stand to obtain a second treated product; preferably, the standing time is 2-3 minutes.

[0030] In any embodiment of this application, in step 2), the centrifugation conditions are 4°C, 15000g, and centrifugation time is 10-15min.

[0031] In any embodiment of this application, in step 3), the anhydrous ethanol is 0.5 to 1 times the amount of the supernatant.

[0032] In any embodiment of this application, in step 3), the separation of the magnetic beads is performed on a magnetic rack.

[0033] In any embodiment of this application, step 5) is repeated multiple times.

[0034] In any embodiment of this application, in step 3), anhydrous ethanol and magnetic bead solution are added to the deep well plate, and subsequent steps 4) to 6) are performed in an automated nucleic acid extractor to achieve semi-automated extraction.

[0035] The third aspect of this application provides the use of the kit described in the first aspect for extracting total RNA from plant tissues.

[0036] The fourth aspect of this application provides the use of the method described in the second aspect for extracting total RNA from plant tissues.

[0037] The fifth aspect of this application provides total RNA from plant tissue, extracted by the method described in the second aspect.

[0038] Compared with the prior art, the beneficial effects of this application are as follows:

[0039] 1. This invention provides a simple, rapid plant tissue RNA extraction protocol based on magnetic beads. It allows for manual operation and easy integration with various automated nucleic acid extraction instruments. It simplifies experimental procedures to the greatest extent possible, ensuring the safety of experimental personnel. It meets the requirements for nucleic acid extraction speed, throughput, and quality. The protocol provided by this invention is simple to operate, has high sample throughput, and offers advantages such as high RNA recovery rate, high purity, and intact fragments.

[0040] 2. This invention improves RNA extraction from plant tissues rich in phenols, quinones, polysaccharides, and secondary metabolites by utilizing Tris-boric acid, EDTA, and potassium acetate. The addition of phenol and chloroform can also remove most of the protein while inhibiting DNA dissolution, thus improving purification efficiency.

[0041] 3. The entire process can be completed within 50 minutes, which reduces the risk of RNA degradation during extraction. Combined with the characteristics of nanomagnetic beads, the operation is simple and more suitable for mechanized and semi-automated operation, resulting in high purity, high integrity and high quality plant tissue RNA. Attached Figure Description

[0042] Figure 1 The image shown is an electrophoresis diagram from an embodiment of this application.

[0043] Figure 2 The image shown is an electrophoresis diagram from the comparative examples of this application. Detailed Implementation

[0044] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0045] The inventors of this application, through extensive research and exploration, discovered a method for rapidly extracting total RNA from plant tissues based on magnetic beads, and completed this application based on this method.

[0046] This application provides a kit for extracting total RNA from plant tissues, comprising one or more of the following raw materials in combination:

[0047] Lysis buffer: including one or more of the following: 2-6M guanidine isothiocyanate (GITC), 0.01-0.3M boric acid, 50-500mM tris(hydroxymethyl)aminomethane-hydrochloric acid, 20-50mM sodium chloride, and 20-50mM disodium ethylenediaminetetraacetate (EDTA·2Na);

[0048] Potassium acetate solution: The solutes include 2.5M potassium acetate and / or 0.5M potassium chloride;

[0049] Magnetic bead solution: includes magnetic beads, which are selected from silanol magnetic beads or carboxyl magnetic beads;

[0050] Protein removal buffer: Solutes include protein denaturants and / or salt ions;

[0051] Nucleic acid elution buffer: The solute includes 0.1% diethyl pyrocarbonate.

[0052] Unless otherwise specified, the solvent in the kit is generally sterile deionized water or DEPC water. Those skilled in the art will recognize that other suitable solvents may also be used in this application.

[0053] The kit provided in this application also includes one or more combinations of wash buffer, phenol, chloroform, and anhydrous ethanol. In one specific embodiment of this application, the wash buffer is an ethanol solution. In some embodiments, the wash buffer is a 75% (v / v) aqueous ethanol solution.

[0054] In the kit provided in this application, the pH of the tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) in the lysis buffer is 7.0-7.5.

[0055] In the kit provided in this application, the pH of potassium chloride in the potassium acetate solution is 4.8-5.2.

[0056] The kit provided in this application utilizes the superparamagnetism of silica-coated magnetic nanospheres to separate RNA from tissue samples under the influence of a liquid salt (such as guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field. In specific embodiments of this application, the magnetic bead solution can be any one of commercially available silanol magnetic bead solutions and carboxyl magnetic bead solutions.

[0057] The reagent kit provided in this application includes a protein denaturing agent in the protein removal buffer comprising one or more combinations of urea, guanidine salt, and proteinase K. In a preferred embodiment of this application, the guanidine salt comprises guanidine isothiocyanate and / or guanidine hydrochloride. The salt ions in the protein removal buffer comprise one or more combinations of LiCl, NaCl, and KCl.

[0058] In some embodiments, the solute in the protein removal buffer includes 2-4 M guanidine isothiocyanate and / or 0.5-2 M LiCl.

[0059] In the kit provided in this application, the volume ratio of phenol to chloroform is 3:2.

[0060] The second aspect of this application provides a method for extracting total RNA from plant tissues, using the aforementioned kit, comprising the following steps:

[0061] 1) Grind the plant tissue into powder, add lysis buffer and potassium acetate solution to obtain the first treatment product;

[0062] 2) Centrifuge the first treated product, take the supernatant, add phenol and chloroform to obtain the second treated product;

[0063] 3) Centrifuge the second-processed material, take the supernatant, add anhydrous ethanol and magnetic bead solution, mix well, and then separate the magnetic beads;

[0064] 4) After mixing the magnetic beads with the protein removal buffer, separate the magnetic beads;

[0065] 5) After mixing the magnetic beads with the rinsing buffer, separate the magnetic beads;

[0066] 6) Elute the magnetic beads with nucleic acid elution buffer to obtain total RNA from plant tissue.

[0067] In the method provided in this application, step 1) refers to grinding plant tissue into powder, adding lysis buffer and potassium acetate solution to obtain the first processed product. The plant tissue is selected from the roots, stems, leaves, flowers, or fruits of plants. The plants are selected from common plant varieties such as tomatoes and soybeans, or medicinal plant varieties such as Elsholtzia ciliata. The extraction amount of plant tissue is 50-100 mg.

[0068] In the method provided in this application, step 1) involves grinding in liquid nitrogen; the grinding is performed in a grinder with a frequency of 45–55 Hz and a grinding time of 15–20 s. Grinding is carried out under liquid nitrogen to protect the RNA from degradation by RNases.

[0069] In the method provided in this application, in step 1), guanidine isothiocyanate in the lysis buffer can rapidly decompose cells and inhibit the release of RNase from cells. Tris-boric acid forms a complex with phenolic compounds through hydrogen bonds, thereby inhibiting the oxidation of phenolic substances and their binding to RNA. Tris-HCl provides a suitable lysis environment, sodium chloride maintains the stability of nucleic acid structure and provides a buffer reaction environment, EDTA inhibits the damage of nucleic acids to nucleic acids by nucleases in the sample during lysis, and NaCl maintains the stability of nucleic acid structure, thus achieving the release of high-quality RNA during lysis.

[0070] In the method provided in this application, step 1) involves using potassium acetate to precipitate polysaccharides, thereby separating RNA from polysaccharide substances.

[0071] In the method provided in this application, in step 1), after adding lysis buffer and potassium acetate solution, the mixture is allowed to stand to obtain the first processed product. In a specific embodiment, the standing time is 2-3 minutes.

[0072] In the method provided in this application, step 2) refers to centrifuging the first processed product, taking the supernatant, and adding phenol and chloroform to obtain the second processed product. The centrifugation conditions are 4°C, 15000g, and a centrifugation time of 2 minutes.

[0073] In the method provided in this application, in step 2), the acidic buffer environment is enhanced by phenol and guanidine isothiocyanate in the lysis system, thereby achieving the separation of RNA from DNA and protein impurities.

[0074] In the method provided in this application, in step 2), phenol and chloroform are added, and the mixture is allowed to stand to obtain a second treated product. In one specific embodiment, the standing time is 2-3 minutes.

[0075] In the method provided in this application, step 3) refers to centrifuging the second processed material, collecting the supernatant, adding anhydrous ethanol and magnetic bead solution, mixing well, and then separating the magnetic beads. The centrifugation conditions are 4°C, 15000g, and a centrifugation time of 10-15 min. Magnetic beads, for example, can be purchased from Meiji Yuhua RNA adsorption magnetic beads (hydroxyl magnetic beads). The magnetic beads are used to adsorb RNA.

[0076] In the method provided in this application, in step 3), the anhydrous ethanol is 0.5 to 1 times the amount of the supernatant; preferably, it can be 0.5 to 1 times, 0.5 to 1 times, 0.5 to 1 times, etc. In a preferred embodiment of this application, the anhydrous ethanol is 0.6 times the amount of the supernatant. Anhydrous ethanol is used to precipitate RNA.

[0077] In the method provided in this application, step 3) involves separating the magnetic beads on a magnetic rack.

[0078] In the method provided in this application, step 4) refers to separating the magnetic beads after mixing them with a protein removal buffer. The separation of the magnetic beads is performed on a magnetic rack. The protein removal buffer is used to remove protein impurities to obtain relatively pure RNA.

[0079] In the method provided in this application, step 5) refers to separating the magnetic beads after mixing them with the rinsing buffer. Step 5) can be repeated multiple times, for example, twice.

[0080] In the method provided in this application, in step 3), when the sample throughput is not less than 32 samples, anhydrous ethanol and nucleic acid solution are added to the deep-well plate. Subsequent steps 4) to 6) are performed in an automated nucleic acid extractor, achieving semi-automated extraction. Those skilled in the art will understand that a conventional commercially available automated nucleic acid extractor can be used. For example, it can be selected from Ausen Auto-Pure 32 or BioHandler Volador-96; other conventional automated nucleic acid extractors can also be used in this application. The deep-well plate can be a deep-well plate well-known to those skilled in the art, such as a 96-well plate. The method provided in this application is simple to operate, has a high sample throughput, and provides RNA extraction with advantages such as high recovery rate, high purity, and intact fragments.

[0081] The third aspect of this application provides the use of the aforementioned kit for extracting total RNA from plant tissues.

[0082] The fourth aspect of this application provides the use of the aforementioned method in extracting total RNA from plant tissues.

[0083] The fifth aspect of this application provides total RNA from plant tissue, which is extracted by the aforementioned method.

[0084] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0085] Specific reagents include:

[0086] The lysis buffer consisted of 3M GITC, 0.2M boric acid, 50mM Tris-HCl pH 7.0-7.5, 30mM sodium chloride, and 40mM EDTA·2Na.

[0087] The protein removal buffer is: 4M GITC, 1M LiCl;

[0088] The potassium acetate solution is: 2.5M potassium acetate, 0.5M potassium chloride, pH 4.8-5.2;

[0089] The rinsing buffer is 75% ethanol (v / v);

[0090] The nucleic acid elution buffer was 0.1% diethyl pyrocarbonate (DEPC), and the mixture was autoclaved for 30 minutes.

[0091] For a throughput of 32 samples, the automated nucleic acid extractor used was the Auto-Pure 32; for a throughput of 64 samples, the automated nucleic acid extractor used was the BioHandler Volador-96.

[0092] Example 1: Rapid extraction of total RNA from Elsholtzia ciliata root tissue samples using magnetic beads.

[0093] 1.1 A method for rapid extraction of total RNA from root tissue samples of Elsholtzia ciliata using magnetic beads

[0094] a. Grind 50-100 mg of Elsholtzia ciliata root tissue into powder under liquid nitrogen, add 600 μL of lysis buffer and mix well to ensure thorough mixing of tissue powder and lysis buffer. Add 80 μL of potassium acetate, mix thoroughly, and let stand at room temperature for 2-3 min;

[0095] b. Centrifuge at 15000g for 2 minutes at 4℃, take the supernatant, add 300μL of phenol, shake well, add 200μL of chloroform, shake well, and let stand at room temperature for 3 minutes.

[0096] c. Centrifuge at 15000g at 4℃ for 15 min, take the supernatant, add 0.6 times the volume of anhydrous ethanol and 10 μL of magnetic bead solution, vortex mix for 30 seconds, adsorb on a magnetic rack for 1 min, and discard the supernatant.

[0097] d. Add 500 μL of protein removal buffer, vortex for 30 seconds, magnetically attach for 1 minute, and discard the supernatant;

[0098] e. Continue adding 500 μL of rinsing buffer, vortex mix for 30 seconds, magnetic rack adsorption for 1 min, discard the supernatant, and repeat twice.

[0099] f. Finally, add 50 μL of nucleic acid elution buffer to elute the nucleic acid;

[0100] g. The extracted nucleic acid extract can be used immediately or stored at -80°C.

[0101] 1.2 A method for rapidly extracting total RNA from root tissue samples of Elsholtzia ciliata using a 32-sample-throughput nucleic acid extractor and magnetic bead method.

[0102] a. Grind 50-100 mg of Elsholtzia ciliata root tissue into powder under liquid nitrogen, add 600 μL of lysis buffer and mix well to ensure thorough mixing of tissue powder and lysis buffer. Add 80 μL of potassium acetate, mix thoroughly, and let stand at room temperature for 2-3 min;

[0103] b. Centrifuge at 15000g for 2 min at 4℃, take the supernatant, add 300μL of phenol, shake well, add 200μL of chloroform, shake well, let stand at room temperature for 3 min, centrifuge at 15000g for 15 min at 4℃.

[0104] c. Add the reagent for extracting nucleic acid with magnetic beads to a 96-well deep plate. Add 0.6 times the volume of anhydrous ethanol and 10 μL of magnetic bead solution to columns 1 and 7. Add 500 μL of protein removal buffer to columns 2 and 8. Add 500 μL of wash buffer to columns 3, 4, 9 and 10. Add 50 μL of nucleic acid elution buffer to columns 5 and 11.

[0105] d. Take the supernatant from step b and add it to columns 1 and 7 of a 96-well deep plate. Place it in an automated nucleic acid extractor and run the program to obtain the total RNA aqueous solution extracted from the root tissue of Elsholtzia ciliata.

[0106] 1.3 A method for rapidly extracting total RNA from Elsholtzia ciliata root tissue samples using a 96-sample-throughput nucleic acid extractor and magnetic bead method.

[0107] a. Grind 50-100 mg of plant tissue into powder under liquid nitrogen, add 600 μL of lysis buffer and mix well to ensure thorough mixing of the tissue powder and lysis buffer. Add 80 μL of potassium acetate, mix thoroughly, and let stand at room temperature for 2-3 minutes;

[0108] b. Centrifuge at 15000g for 2 min at 4℃, take the supernatant, add 300μL of phenol, shake well, add 200μL of chloroform, shake well, let stand at room temperature for 3 min, centrifuge at 15000g for 15 min at 4℃.

[0109] a. Add the reagent for extracting nucleic acid with magnetic beads to a 96-well deep plate. Add 0.6 times the volume of anhydrous ethanol and 10 μL of magnetic bead solution to well 1 of step b. Add 500 μL of protein removal buffer to well 2. Add 500 μL of wash buffer to wells 3 and 4. Add 50 μL of nucleic acid elution buffer to well 5.

[0110] c. Take the supernatant from step b and add it to a 96-well deep plate (No. 1). Place the plate in an automated nucleic acid extractor and run the program to obtain the extracted RNA aqueous solution from Elsholtzia ciliata root tissue.

[0111] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 1. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract the root tissue of Elsholtzia ciliata, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0112] Table 1. Data on the detection of total RNA quality in root tissues of Elsholtzia ciliata.

[0113]

[0114] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0115] Experiment 2 uses the magnetic bead method to rapidly extract total RNA from stem tissue samples of Elsholtzia ciliata.

[0116] 2.1 In this experiment, the reagents and protocol in Example 1.1 were used to extract total RNA from the stem tissue of Elsholtzia ciliata.

[0117] 2.2 In this experiment, the reagents and procedures in Example 1.2 were used to extract total RNA from the stem tissue of Elsholtzia ciliata.

[0118] 2.3 In this experiment, the reagents and procedures in Example 1.3 were used to extract total RNA from the stem tissue of Elsholtzia ciliata.

[0119] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 2. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract total RNA from the stem tissue of Elsholtzia ciliata, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0120] Table 2. Data on total RNA quality from stem tissues of Elsholtzia ciliata.

[0121]

[0122] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0123] Experiment 3 used the magnetic bead method to rapidly extract total RNA from Elsholtzia ciliata leaf tissue samples.

[0124] 3.1 In this experiment, the reagents and protocol in Experiment 1.1 were used to extract total RNA from the leaf tissue of Elsholtzia ciliata.

[0125] 3.2 In this experiment, the reagents and procedures in Example 1.2 were used to extract total RNA from Elsholtzia ciliata leaf tissue.

[0126] 3.3 In this experiment, the reagents and procedures in Example 1.3 were used to extract total RNA from Elsholtzia ciliata leaf tissue.

[0127] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 3. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract total RNA from Elsholtzia ciliata leaf tissue, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0128] Table 3. Data on the detection of total RNA quality in Elsholtzia ciliata leaf tissue.

[0129]

[0130] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0131] Experiment 4 uses magnetic beads to rapidly extract total RNA from tissue samples of Elsholtzia ciliata flowers.

[0132] 4.1 In this experiment, the reagents and protocol in Example 1.1 were used to extract total RNA from the tissue of Elsholtzia ciliata flower.

[0133] 4.2 In this experiment, the reagents and procedures in Example 1.2 were used to extract total RNA from Elsholtzia ciliata flower tissue.

[0134] 4.3 In this experiment, the reagents and procedures in Example 1.3 were used to extract total RNA from Elsholtzia ciliata flower tissue.

[0135] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 4. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract RNA from Elsholtzia splendens flower tissue, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0136] Table 4. Data on the detection of total RNA quality in Elsholtzia ciliata flower tissues.

[0137]

[0138] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0139] Experiment 5 uses magnetic beads to rapidly extract total RNA from tomato fruit tissue samples.

[0140] 5.1 This experiment used the reagents and protocol in Example 1.1 to extract total RNA from tomato fruit tissue.

[0141] 5.2 In this experiment, the reagents and procedures in Example 1.2 were used to extract total RNA from tomato fruit tissue.

[0142] 5.3 In this experiment, the reagents and procedures in Example 1.3 were used to extract total RNA from tomato fruit tissue.

[0143] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 5. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract RNA from tomato fruit tissue, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0144] Table 5. Data on total RNA quality from tomato fruit tissue.

[0145]

[0146] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0147] Experiment 6 uses magnetic beads to rapidly extract total RNA from soybean seed tissue samples.

[0148] 6.1 In this experiment, the reagents and protocol in Example 1.1 were used to extract total RNA from soybean seed tissue.

[0149] 6.2 In this experiment, the reagents and procedures in Example 1.2 were used to extract total RNA from soybean seed tissue.

[0150] 6.3 In this experiment, the reagents and procedures in Example 1.3 were used to extract total RNA from soybean seed tissue.

[0151] The concentration and purity of nucleic acids were measured using Nanodrop, and the integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the experimental method are shown in Table 6. Figure 1 Results of nucleic acid extraction via agarose gel electrophoresis. The results show that the method of the present invention can completely extract RNA from soybean seed tissue, and the nucleic acid yield, nucleic acid purity, and nucleic acid agarose gel electrophoresis results are good.

[0152] Table 6. Data on total RNA quality from soybean seed tissues.

[0153]

[0154] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0155] Comparative Example 1

[0156] RNA was extracted from the leaves of Elsholtzia ciliata in this comparative example.

[0157] All plastic centrifuge tubes and grinding tubes used in the following experimental methods were sterilized at 121℃ for 30 minutes and then dried for later use; pipette tips (sterile and enzyme-free, purchased from Axygen); pipettes and experimental workbenches were irradiated with ultraviolet light for 30 minutes and wiped with RNAzap.

[0158] Specific reagents include:

[0159] The lysis buffer consisted of: 1M GITC, 0.2M boric acid, 50mM Tris-HCl pH 8.0, 30mM sodium chloride, and 40mM EDTA·2Na.

[0160] The protein removal buffer is: 4M GITC, 1M LiCl;

[0161] The potassium acetate solution is: 2.5M potassium acetate, 0.5M potassium chloride, pH 4.8-5.2;

[0162] The rinsing buffer is 75% ethanol (v / v);

[0163] The nucleic acid elution buffer was 0.1% diethyl pyrocarbonate (DEPC), and the mixture was autoclaved for 30 minutes.

[0164] For a throughput of 32 samples, the automated nucleic acid extractor used was the Auto-Pure 32; for a throughput of 64 samples, the automated nucleic acid extractor used was the BioHandler Volador-96.

[0165] This experiment used the protocol in Experiment 1.1 to extract total RNA from Elsholtzia ciliata leaf tissue.

[0166] Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose gel electrophoresis. The purity and yield of nucleic acids extracted by the comparative method are shown in Table 7. Figure 2 Results of nucleic acid extraction via agarose gel electrophoresis. The results showed that the RNA and nucleic acid yields from the Elsholtzia ciliata leaf tissue extracted using this method were low, and the nucleic acid agarose gel electrophoresis results indicated DNA contamination.

[0167] Table 7. Data on total RNA quality analysis of Elsholtzia ciliata leaf tissue.

[0168]

[0169] Note: Each extraction method was repeated three times, and the quality control results were averaged.

[0170] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. A kit for total RNA extraction from plant tissues, comprising the following raw material combination: Lysis buffer: a combination of 2-6 M guanidine isothiocyanate, 0.01-0.3 M boric acid, 50-500 mM tris(hydroxymethyl)aminomethane-hydrochloric acid, 20-50 mM sodium chloride, and 20-50 mM disodium ethylenediaminetetraacetate; Potassium acetate solution: The solute is selected from 2.5M potassium acetate and 0.5M potassium chloride, and the pH of potassium chloride in the potassium acetate solution is 4.8-5.2; Magnetic bead solution: comprising magnetic beads, wherein the magnetic beads are selected from silanol magnetic beads or carboxyl magnetic beads; Protein removal buffer: Solutes selected from 2-4M guanidine isothiocyanate and 0.5-2M LiCl; Nucleic acid elution buffer: The solute includes 0.1% diethyl pyrocarbonate; It also includes a rinsing buffer, phenol, chloroform, and anhydrous ethanol, wherein the rinsing buffer is an ethanol solution.

2. The kit according to claim 1, characterized in that, The pH of the lysis buffer solution containing tris(hydroxymethyl)aminomethane-hydrochloric acid is 7.0-7.

5. And / or, the volume ratio of phenol to chloroform is 3:

2.

3. A method for extracting total RNA from plant tissues, using the kit described in claim 1 or 2, comprising the following steps: 1) Grind the plant tissue into powder, add lysis buffer and potassium acetate solution to obtain the first treatment product; 2) Centrifuge the first treated product, take the supernatant, add phenol and chloroform to obtain the second treated product; 3) Centrifuge the second-processed material, take the supernatant, add anhydrous ethanol and magnetic bead solution, mix well, and then separate the magnetic beads; 4) After mixing the magnetic beads with the protein removal buffer, separate the magnetic beads; 5) After mixing the magnetic beads with the rinsing buffer, separate the magnetic beads; 6) Elute the magnetic beads with nucleic acid elution buffer to obtain total RNA from plant tissue.

4. The method as described in claim 3, characterized in that, Includes a combination of one or more of the following features: a1) In step 1), the plant tissue is selected from the root, stem, leaf, flower, or fruit tissue of a plant; a2) In step 1), the grinding is carried out in liquid nitrogen; the grinding is carried out in a grinding machine with a frequency of 45-55Hz and a grinding time of 15-20s; a3) In step 1), after adding lysis buffer and potassium acetate solution, the first treated product is obtained by standing. a4) In step 2), the centrifugation conditions are 4℃, 15000g, and centrifugation time is 2-5min; a5) In step 2), after adding phenol and chloroform, let it stand to obtain the second treated product. The standing time is 2-3 minutes. a6) In step 3), the centrifugation conditions are 4℃, 15000g, and centrifugation time is 10-15min; a7) In step 3), the anhydrous ethanol is 0.5 to 1 times the amount of the supernatant; In step 3) of a8), the separation of the magnetic beads is performed on a magnetic rack; a9) Step 5) Repeat multiple times.

5. The method as described in claim 3, characterized in that, In step 3), anhydrous ethanol and magnetic bead solution are added to the deep well plate. Subsequent steps 4) to 6) are performed in an automated nucleic acid extractor to achieve semi-automated extraction.