CN116004613A

CN116004613A - Method for rapidly extracting total RNA of plant tissues based on magnetic bead method

Info

Publication number: CN116004613A
Application number: CN202310191557.7A
Authority: CN
Inventors: 李桃; 董亚晨; 陈晓翠
Original assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Current assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Priority date: 2023-03-02
Filing date: 2023-03-02
Publication date: 2023-04-25

Abstract

The application relates to the technical field of RNA extraction, in particular to a method for rapidly extracting total RNA of plant tissues based on a magnetic bead method. The method can be suitable for extracting total RNA of plant tissues such as roots, stems, leaves, flowers, fruits and the like, can obtain high-quality sample total RNA through a simple and rapid experimental process, is suitable for quantitative PCR and transcriptome sequencing, and has a high application value.

Description

Method for rapidly extracting total RNA of plant tissues based on magnetic bead method

Technical Field

The application relates to the technical field of RNA extraction, in particular to a method for rapidly extracting total RNA of plant tissues based on a magnetic bead method.

Background

Extraction of RNA from plant tissues is a necessary prerequisite for research in plant molecular biology. For Northern hybridization analysis, purification of mRNA for in vitro translation or establishment of cDNA library, molecular biology research such as RT-PCR and differential analysis, high quality RNA is required. Therefore, the extraction of RNA with high purity and good integrity from plant tissues is a key point for successful research.

In most plant tissues, the activity of RNase is high, which is rich in phenolic compounds, polysaccharides and some secondary metabolites which are not yet confirmed, and the RNA in the plant tissues cannot be effectively separated and purified. When plant tissue is ground, and cells are broken, these substances interact with RNA. The phenolic compound is irreversibly bound to RNA after being oxidized to quinone; the polysaccharide forms insoluble jelly, and is coprecipitated with RNA; terpenoids and RNase cause chemical degradation and enzymatic hydrolysis of RNA, respectively, resulting in low yields and poor quality of the obtained RNA. For such plant tissue samples, it is difficult to extract their RNA by conventional RNA extraction methods (e.g., guanidine method, phenol method, hexadecyl trimethyl amine bromide method, etc.).

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a method for rapidly extracting total RNA from plant tissue based on a magnetic bead method, which is used for solving the problems of complicated steps and low extraction rate of multiple types of reagents in the prior art. The method can be suitable for the scheme of extracting total RNA of plant tissues such as roots, stems, leaves, flowers, fruits and the like, can obtain the sample total RNA with higher quality through a simpler and faster experimental process, is suitable for quantitative PCR and transcriptome sequencing, and has higher application value.

To achieve the above and other related objects, a first aspect of the present application provides a kit for total RNA extraction from plant tissue, comprising one or more of the following raw materials in combination:

lysis buffer: comprises one or more of 2-6M guanidine isothiocyanate, 0.01-0.3M boric acid, 50-500mM tris-hydrochloric acid, 20-50mM sodium chloride and 20-50mM disodium edetate;

potassium acetate solution: the solute comprises 2.5M potassium acetate and/or 0.5M potassium chloride;

magnetic bead solution: comprises magnetic beads which are selected from silicon hydroxyl magnetic beads or carboxyl magnetic beads;

deproteinization buffer: solutes include protein denaturants and/or salt ions;

nucleic acid eluate: the solute included 0.1% diethyl pyrocarbonate.

In any embodiment of the present application, the kit further comprises a combination of one or more of a rinse buffer, phenol, chloroform, absolute ethanol; preferably, the rinsing buffer is an ethanol solution.

In any embodiment of the present application, the pH of the tris-hcl in the lysis buffer is 7.0-7.5.

In any embodiment of the present application, the pH of the potassium chloride in the potassium acetate solution is 4.8-5.2.

In any embodiment of the present application, the protein denaturing agent in the deproteinization buffer comprises a combination of one or more of urea, guanidinium, and proteinase K; preferably, the guanidine salt comprises guanidine isothiocyanate and/or guanidine hydrochloride.

In any embodiment of the present application, the salt ions in the deproteinization buffer comprise a combination of one or more of LiCl, naCl, and KCl.

In any embodiment of the present application, the volume ratio of phenol to chloroform is 3:2.

in any embodiment of the present application, the solute of the deproteinization buffer comprises 2-4M guanidine isothiocyanate and/or 0.5-2M LiCl.

In a second aspect, the present application provides a method for extracting total RNA from plant tissue, which is performed using the kit according to the first aspect, and comprises the following steps:

1) Grinding plant tissues into powder, and adding a lysis buffer and a potassium acetate solution to obtain a first treated substance;

2) Centrifuging the first treated matter, taking supernatant, and adding phenol and chloroform to obtain a second treated matter;

3) Centrifuging the second treated matter, taking supernatant, adding absolute ethyl alcohol and magnetic bead solution, uniformly mixing, and separating magnetic beads;

4) Uniformly mixing the magnetic beads with deproteinized buffer solution, and separating the magnetic beads;

5) Uniformly mixing the magnetic beads with a rinsing buffer solution, and separating the magnetic beads;

6) Eluting the magnetic beads with nucleic acid eluent to obtain total RNA of plant tissues.

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

In any embodiment of the present application, in step 1), the milling is performed in liquid nitrogen; the grinding is carried out in a grinding instrument, the frequency of the grinding instrument is 45-55 Hz, and the grinding time is 15-20 s.

In any embodiment of the present application, in step 1), adding a lysis buffer and a potassium acetate solution, and standing to obtain a first treated substance; preferably, the first standing time is 2-3min.

In any embodiment of the present application, in step 2), the centrifugation conditions are 4 ℃,15000g, and the centrifugation time is 2-5min.

In any embodiment of the present application, in step 2), phenol and chloroform are added and then left to stand to obtain a second treated product; preferably, the rest time is 2-3min.

In any embodiment of the present application, in step 2), the centrifugation conditions are 4 ℃,15000g, and the centrifugation time is 10-15min.

In any embodiment of the present application, in step 3), the absolute ethanol is 0.5 to 1 times the supernatant.

In any embodiment of the present application, in step 3), the separating the magnetic beads is performed on a magnetic rack.

In any embodiment of the present application, step 5) is repeated a plurality of times.

In any embodiment of the present application, in step 3), the anhydrous ethanol and the magnetic bead solution are added into a deep well plate, and the subsequent steps 4) to 6) are performed in an automatic nucleic acid extractor, thereby realizing semi-automatic extraction.

In a third aspect, the present application provides the use of a kit according to the first aspect for extracting total RNA from plant tissue.

In a fourth aspect the present application provides the use of the method of the second aspect for extracting total RNA from plant tissue.

In a fifth aspect, the present application provides total RNA from plant tissue extracted by the method of the second aspect.

Compared with the prior art, the beneficial effects of this application are:

1. the invention provides a magnetic bead method-based rapid plant tissue RNA scheme with simple operation, which can realize manual operation and is convenient for integrating various automatic nucleic acid extractors. The experimental operation steps are simplified to the greatest extent, and the safety of experimental staff is ensured. Meets the requirements of nucleic acid extraction speed, flux and nucleic acid quality. The scheme provided by the invention is simple to operate, high in sample flux, high in recovery rate, high in purity, complete in fragment and the like.

2. The invention improves the extraction of plant tissue RNA rich in phenols, quinones, polysaccharide, secondary metabolites and the like by utilizing Tris-boric acid, EDTA and potassium acetate. The addition of phenol and chloroform also can inhibit DNA dissolution while removing most of the proteins, improving purification efficiency.

3. The whole process can be completed within 50 minutes, the operation time is short, and the risk of RNA degradation in the extraction process is reduced; the characteristic of the nanometer magnetic beads is combined, so that the operation process is simple, and the method is more suitable for mechanized and semi-automatic operation, and the plant tissue RNA with high purity, high integrity and high quality is obtained.

Drawings

Fig. 1 shows an electrophoresis diagram in an embodiment of the present application.

FIG. 2 shows an electrophoresis pattern in comparative example of the present application.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventor of the application finds a method for rapidly extracting total RNA of plant tissues based on a magnetic bead method through a great deal of research and study, and completes the application on the basis.

In one aspect, the application provides a kit for total RNA extraction of plant tissues, which comprises one or more of the following raw materials:

lysis buffer: comprises one or more of 2-6M Guanidine Isothiocyanate (GITC), 0.01-0.3M boric acid, 50-500mM tris-hydrochloric acid, 20-50mM sodium chloride and 20-50mM disodium ethylenediamine tetraacetate (EDTA.2Na);

deproteinization buffer: solutes include protein denaturants and/or salt ions;

nucleic acid eluate: the solute included 0.1% diethyl pyrocarbonate.

The solvent in the kit is typically sterile deionized water or DEPC water unless otherwise specified. It should be well known to those skilled in the art that other suitable solvents may be used in the present application.

The kit further comprises one or more of a rinsing buffer, phenol, chloroform and absolute ethyl alcohol. In one embodiment of the present application, the rinse buffer is an ethanol solution. In some embodiments, the rinse buffer is a 75% (V/V) aqueous ethanol solution.

In the kit provided by the application, the pH of the Tris-hydrochloric acid (Tris-HCl) in the lysis buffer is 7.0-7.5.

In the kit provided by the application, the pH of potassium chloride in the potassium acetate solution is 4.8-5.2.

In the kit provided by the application, the magnetic bead solution can separate RNA from a tissue sample under the action of chaotropic salts (such as guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field by utilizing the superparamagnetism of the nano magnetic microsphere coated with silicon dioxide. In the specific embodiment of the present application, the magnetic bead solution may be any one of a commercially available silicon hydroxyl magnetic bead solution and a carboxyl magnetic bead solution.

In the kit provided by the application, the protein denaturing agent in the deproteinization buffer comprises one or more of urea, guanidine salt and proteinase K. In a preferred embodiment of the present application, the guanidinium salt comprises guanidinium isothiocyanate and/or guanidinium hydrochloride. The salt ions in the deproteinization buffer include a combination of one or more of LiCl, naCl, and KCl.

In some embodiments, the solute of the deproteinization buffer comprises 2-4M guanidine isothiocyanate and/or 0.5-2M LiCl.

In the kit provided by the application, the volume ratio of phenol to chloroform is 3:2.

in a second aspect, the present application provides a method for extracting total RNA from plant tissues, which is performed by using the aforementioned kit, and comprises the following steps:

In the method provided by the application, the step 1) is to grind plant tissues into powder, and add a lysis buffer and a potassium acetate solution to obtain a first treated substance. Wherein the plant tissue is selected from the group consisting of root, stem, leaf, flower or fruit tissue of a plant. The plant is selected from conventional plant varieties such as tomato, soybean, etc., or medicinal plant varieties such as elsholtzia pachyrhizi. The extraction amount of plant tissue is 50-100mg.

In the method provided by the application, in the step 1), the grinding is carried out in liquid nitrogen; the grinding is carried out in a grinding instrument, the frequency of the grinding instrument is 45-55 Hz, and the grinding time is 15-20 s. Milling is performed under liquid nitrogen, protecting the RNA from rnase degradation.

In the method provided by the application, in the step 1), the guanidine isothiocyanate in the lysis buffer can rapidly decompose cells and inhibit release of RNase by the cells. Tris-boric acid forms a complex with phenolic compounds through hydrogen bonds, thereby inhibiting oxidation of phenolic substances and combination of the phenolic substances with RNA, tris-HCl provides a proper cracking environment, sodium chloride maintains stability of a nucleic acid structure and provides a buffer reaction environment, EDTA inhibits nuclease in a sample from damaging nucleic acid in a cracking process and NaCl maintains stability of the nucleic acid structure, and high-quality RNA is released in the cracking process.

In the method provided by the application, in the step 1), potassium acetate is utilized to precipitate polysaccharide, so that RNA and polysaccharide substances are separated.

In the method provided by the application, in the step 1), a first treated substance is obtained by adding a lysis buffer solution and a potassium acetate solution and then standing. In one embodiment, the resting time is 2-3 minutes.

In the method provided by the application, the step 2) is to centrifuge the first treatment substance, take the supernatant, and add phenol and chloroform to obtain the second treatment substance. Wherein, the centrifugation condition is 4 ℃,15000g and the centrifugation time is 2min.

In the method provided by the application, in the step 2), the separation of RNA, DNA and protein impurities is realized by improving the acidic buffer environment through phenol and guanidine isothiocyanate in a cracking system.

In the method provided by the application, in the step 2), phenol and chloroform are added and then the mixture is stood still to obtain a second treated substance. In one embodiment, the resting time is 2-3 minutes.

In the method provided by the application, the step 3) is to centrifuge the second treated substance, take the supernatant, add absolute ethyl alcohol and magnetic bead solution, mix uniformly and then separate the magnetic beads. Wherein the centrifugation condition is 4deg.C, 15000g, and the centrifugation time is 10-15min. The magnetic beads are commercially available, for example, from Megaku Yu Hua RNA adsorption magnetic beads (hydroxyl magnetic beads). The magnetic beads are used to adsorb RNA.

In the method provided by the application, in the step 3), the absolute ethyl alcohol is 0.5-1 times of the supernatant; preferably, it may be 0.5 to 1 times, etc. In a preferred embodiment of the present application, absolute ethanol is 0.6 times the supernatant. Absolute ethanol was used to precipitate RNA.

In the method provided by the application, in the step 3), the magnetic beads are separated on a magnetic frame.

In the method provided by the application, the step 4) refers to the step of uniformly mixing the magnetic beads with deproteinized buffer solution and then separating the magnetic beads. Wherein, the separation of the magnetic beads is carried out on a magnetic frame. The deproteinized buffer is used to remove protein impurities to obtain purer RNA.

In the method provided by the application, the step 5) refers to the step of uniformly mixing the magnetic beads with a rinsing buffer solution and then separating the magnetic beads. Step 5) is repeated a number of times, for example 2 times.

In the method provided by the application, in the step 3), when the sample flux is not less than 32, absolute ethyl alcohol and a nucleic acid solution are added into a deep hole plate, and the subsequent steps 4) to 6) are carried out in an automatic nucleic acid extraction instrument, so that semi-automatic extraction is realized. It will be appreciated by those skilled in the art that the automated nucleic acid extractor may be selected from conventional, commercially available automated nucleic acid extractors. For example, it may be selected from Orient Auto-Pure 32 or BioHandler Volador-96, and other conventional automated nucleic acid extractors may be used in the present application. The deep well plate may be a deep well plate well known to those skilled in the art, for example, a 96 deep well plate. The method provided by the application is simple to operate, high in sample flux, high in recovery rate, high in purity, complete in fragment and the like.

In a third aspect, the present application provides the use of the aforementioned kit for extracting total RNA from plant tissues.

In a fourth aspect, the present application provides the use of the aforementioned method for extracting total RNA from plant tissue.

In a fifth aspect, the present application provides total RNA from plant tissue extracted by the method described above.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

The specific reagents comprise:

the lysis buffer was: 3M GITC, 0.2M boric acid, 50mM Tris-HCl pH 7.0-7.5, 30mM sodium chloride, and 40mM EDTA.2Na.

The deproteinizing buffer is: 4M GITC, 1M LiCl;

the potassium acetate solution is: 2.5M potassium acetate, 0.5M potassium chloride pH 4.8-5.2;

the rinse buffer was: 75% ethanol (V/V);

the nucleic acid eluent is: 0.1% diethyl pyrocarbonate (DEPC), and autoclaving for 30 min.

The automatic nucleic acid extractor used was Orient Auto-Pure 32 at 32 sample flows and BioHandler Volador-96 at 64 sample flows.

Example 1 rapid extraction of Total RNA from Elsholtzia Calpex root tissue samples Using the magnetic bead method

1.1 method for rapidly extracting total RNA from Elsholtzia recta root tissue samples by using magnetic bead method

a. Grinding 50-100mg of Elsholtzia ciliata root tissue into powder under liquid nitrogen, adding 600 μl of lysis buffer, and mixing to make tissue powder and lysis solution fully and uniformly. Adding 80 μL potassium acetate, mixing, standing at room temperature for 2-3min;

b.4 ℃,15000g, centrifuging for 2min, taking supernatant, adding 300 mu L of phenol, shaking up, adding 200 mu L of chloroform, shaking up, and standing for 3min at room temperature;

c.4 ℃,15000g, centrifuging for 15min, taking supernatant, adding absolute ethyl alcohol with the volume of 0.6 times that of the supernatant and 10 mu L of magnetic bead solution, uniformly mixing for 30 seconds by vortex, adsorbing on a magnetic rack for 1min, and discarding the supernatant;

d. adding 500 mu L deproteinized buffer, vortex mixing for 30 seconds, adsorbing for 1min by a magnetic rack, and discarding the supernatant;

e. continuously adding 500 mu L of rinsing buffer solution, vortex mixing for 30 seconds, adsorbing for 1min by a magnetic rack, discarding the supernatant, and repeating for 2 times;

f. finally adding 50 mu L of nucleic acid eluent to elute nucleic acid;

g. the extracted nucleic acid extract can be used immediately or stored at-80 ℃.

1.2 method for rapidly extracting total RNA from Elsholtzia spatholobus stem tissue samples by using 32 sample flux nucleic acid extractor and adopting magnetic bead method

b.4 ℃,15000g, centrifuging for 2min, taking supernatant, adding 300 mu L of phenol, shaking uniformly, adding 200 mu L of chloroform, shaking uniformly, standing at room temperature for 3min,4 ℃,15000g, centrifuging for 15min;

c. adding reagents for extracting nucleic acid from magnetic beads into a 96-well deep hole plate, adding 0.6 times volume of absolute ethyl alcohol and 10 mu L of magnetic bead solution into the supernatant obtained in the step b to the columns 1 and 7, adding 500 mu L of deproteinizing buffer to the columns 2 and 8, adding 500 mu L of rinsing buffer to the columns 3, 4, 9 and 10, and adding 50 mu L of nucleic acid eluent to the columns 5 and 11;

d. adding the supernatant obtained in step b into the 1 st and 7 th columns of 96-hole deep pore plates, placing into an automatic nucleic acid extractor, and running a program to obtain an extracted total RNA aqueous solution of the elsholtzia dens root tissues.

1.3 method for rapidly extracting total RNA from Elsholtzia spatholobus stem tissue samples by using 96 sample flux nucleic acid extractor and adopting magnetic bead method

a. Grinding 50-100mg plant tissue into powder under liquid nitrogen, adding 600 μl of lysis buffer, and mixing to make tissue powder and lysis solution fully mix. Adding 80 μL potassium acetate, mixing, standing at room temperature for 2-3min;

a. adding a reagent for extracting nucleic acid from magnetic beads into a 96-well deep pore plate, adding 0.6 times volume of absolute ethyl alcohol and 10 mu L of magnetic bead solution of the supernatant obtained in the step b into a pore plate No. 1, adding 500 mu L of deproteinizing buffer solution into a pore plate No. 2, adding 500 mu L of rinsing buffer solution into pore plates No. 3 and 4, and adding 50 mu L of nucleic acid eluent into a pore plate No. 5;

c. and b, adding the supernatant obtained in the step b into a No. 1 96-well deep hole plate, putting the plate into an automatic nucleic acid extractor, and running a program to obtain an extracted elsholtzia dens root tissue RNA aqueous solution.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 1. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract the elsholtzia ciliata root tissue, and the nucleic acid yield, the nucleic acid purity and the nucleic acid agarose gel electrophoresis result are good.

TABLE 1 detection data for total RNA quality of Elsholtzia ciliata root tissue

Note that: three repetitions are set for each extraction method, and the quality inspection results are averaged.

Experimental example 2 rapid extraction of Total RNA from Elsholtzia Calpex stem tissue sample by magnetic bead method

2.1 the experiment uses the reagent and the scheme in experimental example 1.1 to extract the total RNA of the stem tissue of the elsholtzia julibrissin

2.2 this experiment was performed using the reagents and protocol of example 1.2 to extract total RNA from stem tissue of Elsholtzia dens.

2.3 the experiment was performed using the reagents and protocol of example 1.3 to extract total RNA from stem tissue of Elsholtzia ciliata.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 2. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract total RNA of the elsholtzia ciliata stem tissue, and has better nucleic acid yield, nucleic acid purity and nucleic acid agarose gel electrophoresis result.

TABLE 2 detection data for total RNA quality of Elsholtzia ciliata stem tissue

Experimental example 3 rapid extraction of Total RNA from Elsholtzia Density leaf tissue samples Using the magnetic bead method

3.1 the experiment adopts the reagent and the scheme in experimental example 1.1 to extract the total RNA of the leaf tissue of the elsholtzia julibrissin

3.2 the experiment was performed using the reagents and protocol of example 1.2 to extract total RNA from leaf tissue of Elsholtzia ciliata.

3.3 this experiment was performed using the reagents and protocol of example 1.3 to extract total RNA from leaf tissue of Elsholtzia dens.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 3. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract total RNA of the elsholtzia ciliata leaf tissue, and has better nucleic acid yield, nucleic acid purity and nucleic acid agarose gel electrophoresis result.

TABLE 3 detection data for Total RNA quality of Elsholtzia leaf tissue

Experimental example 4 rapid extraction of Total RNA from Elsholtzia Calpex tissue sample by magnetic bead method

4.1 the experiment adopts the reagent and the scheme in experimental example 1.1 to extract the total RNA of the elsholtzia densa tissues

4.2 the experiment was performed using the reagents and protocol of example 1.2 to extract total RNA from the tissue of Elsholtzia pachyrhizi.

4.3 the experiment was performed using the reagents and protocol of example 1.3 to extract total RNA from the tissue of Elsholtzia pachyrhizi.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 4. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract the tissue RNA of the elsholtzia ciliata, and the nucleic acid yield, the nucleic acid purity and the nucleic acid agarose gel electrophoresis result are good.

TABLE 4 detection data for total RNA quality of Elsholtzia ciliata tissue

Experimental example 5 rapid extraction of Total RNA from tomato fruit tissue samples Using the magnetic bead method

5.1 this experiment was performed using the reagents and protocol of Experimental example 1.1 to extract total RNA from tomato fruit tissue

5.2 this experiment was performed using the reagents and protocol of example 1.2 to extract total RNA from tomato fruit tissue.

5.3 this experiment was performed using the reagents and protocol of example 1.3 to extract total RNA from tomato fruit tissue.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 5. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract RNA of tomato fruit tissues, and the nucleic acid yield, the nucleic acid purity and the nucleic acid agarose gel electrophoresis result are good.

TABLE 5 detection data for total RNA quality of tomato fruit tissue

Experimental example 6 rapid extraction of Total RNA from soybean seed tissue samples Using the magnetic bead method

6.1 this experiment uses the reagents and protocol of Experimental example 1.1 to extract total RNA from soybean seed tissue

6.2 this experiment was performed to extract total RNA from soybean seed tissue using the reagents and protocol of example 1.2.

6.3 this experiment was performed to extract total RNA from soybean seed tissue using the reagents and protocol of example 1.3.

Nucleic acid concentration and purity were measured using Nanodrop, and integrity was detected by 1.5% agarose electrophoresis; the purity and yield of the nucleic acid extracted by the experimental example method are shown in Table 6. FIG. 1 shows the result of agarose gel electrophoresis of nucleic acid extracted from the nucleic acid. The result shows that the method can completely extract soybean seed tissue RNA, and the nucleic acid yield, the nucleic acid purity and the nucleic acid agarose gel electrophoresis result are good.

TABLE 6 data for the detection of total RNA quality of soybean seed tissue

Comparative example 1

RNA was extracted using Elsholtzia clathrata leaves as an example in this comparative example.

The plastic centrifuge tube and the grinding tube used in the following experimental method are sterilized at 121 ℃ for 30min and then dried for standby; pipetting robbery (sterile enzyme free, purchased from axygen); both the pipetting and the laboratory bench were irradiated with ultraviolet radiation for 30min and wiped with RNAzap.

The specific reagents comprise:

the lysis buffer was: 1M GITC, 0.2M boric acid, 50mM Tris-HCl pH 8.0, 30mM sodium chloride, and 40mM EDTA.2Na;

the deproteinizing buffer is: 4M GITC, 1M LiCl;

the rinse buffer was: 75% ethanol (V/V);

The experiment adopts the scheme in experimental example 1.1 to extract total RNA of the elsholtzia clathrata leaf tissue.

The nucleic acid concentration and purity were measured using Nanodrop, the integrity was checked by 1.5% agarose electrophoresis, and the purity and yield of the nucleic acid extracted by the comparative method are shown in Table 7. FIG. 2 shows the result of agarose gel electrophoresis of nucleic acid extraction. The result shows that the RNA nucleic acid of the elsholtzia ciliata leaf tissue extracted by the scheme has lower yield, and the result of the agarose gel electrophoresis of the nucleic acid shows that DNA pollution exists.

TABLE 7 detection data for total RNA quality of Elsholtzia ciliata leaf tissue

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims

1. A kit for total RNA extraction from plant tissue, comprising a combination of one or more of the following raw materials:

deproteinization buffer: solutes include protein denaturants and/or salt ions;

nucleic acid eluate: the solute included 0.1% diethyl pyrocarbonate.

2. The kit of claim 1, further comprising a combination of one or more of a rinse buffer, phenol, chloroform, absolute ethanol; preferably, the rinsing buffer is an ethanol solution.

3. The kit of claim 2, wherein the pH of tris-hcl in the lysis buffer is 7.0-7.5;

and/or the pH of potassium chloride in the potassium acetate solution is 4.8-5.2;

and/or the protein denaturing agent in the deproteinization buffer comprises a combination of one or more of urea, guanidinium, and proteinase K; preferably, the guanidine salt comprises guanidine isothiocyanate and/or guanidine hydrochloride;

and/or, the salt ions in the deproteinization buffer comprise one or more combinations of LiCl, naCl, and KCl;

and/or, the volume ratio of the phenol to the chloroform is 3:2.

4. the kit of claim 3, wherein the solute of the deproteinization buffer comprises 2-4M guanidine isothiocyanate and/or 0.5-2M LiCl.

5. A method for extracting total RNA from plant tissue using the kit of any one of claims 1 to 4, comprising the steps of:

6. The method of claim 5, comprising a combination of one or more of the following features:

a1 In step 1), the plant tissue is selected from the group consisting of root, stem, leaf, flower or fruit tissue of a plant;

a2 In step 1), the grinding is performed in liquid nitrogen; the grinding is carried out in a grinding instrument, the frequency of the grinding instrument is 45-55 Hz, and the grinding time is 15-20 s;

a3 In step 1), adding a lysis buffer solution and a potassium acetate solution, and standing to obtain a first treated substance; preferably, the first standing time is 2-3min;

a4 In step 2), the centrifugation condition is 4 ℃,15000g, and the centrifugation time is 2-5min;

a5 In step 2), phenol and chloroform are added and then the mixture is stood still to obtain a second treated substance; preferably, the standing time is 2-3min;

a6 In step 3), the centrifugation condition is 4 ℃,15000g, and the centrifugation time is 10-15min;

a7 In the step 3), the absolute ethyl alcohol is 0.5 to 1 time of the supernatant;

a8 In step 3), the separation of the magnetic beads is performed on a magnetic frame;

a9 Step 5) is repeated a plurality of times.

7. The method of claim 5, wherein in step 3), anhydrous ethanol and a magnetic bead solution are added to a deep well plate, and subsequent steps 4) to 6) are performed in an automatic nucleic acid extractor to achieve semi-automatic extraction.

8. Use of a kit according to any one of claims 1 to 4 for extracting total RNA from plant tissue.

9. Use of the method according to any one of claims 5 to 7 for extracting total RNA from plant tissue.

10. Total RNA of plant tissue extracted by the method of any one of claims 5 to 7.