Kit and method for extracting plant RNA based on magnetic beads

Abstract

The invention discloses a kit and a method for extracting plant RNA based on magnetic beads, and belongs to the technical field of plant RNA extraction. The kit comprises a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a DNaseI reaction solution, a second washing solution and an eluent, wherein the lysis solution is prepared from the following components: 10 to 300 mM of sodium tetraborate, 1 to 200 mM of EDTA (Ethylene Diamine Tetraacetic Acid), 0.5 to 15 percent by mass of hexadecyl trimethyl ammonium bromide, 0.5 to 15 percent by mass of lauryl sodium sulfate, 0.5 to 15 percent by mass of sodium dodecyl sulfate, 1 to 10 percent by mass of triton X-100, 1 to 10 percent by mass of Tween 80 and 0.1 to 3 M of sodium chloride; and 10 to 300 mM of tris (hydroxymethyl) aminomethane. By adopting the kit provided by the invention, the plant RNA with higher purity, higher concentration and better integrity can be extracted.

Description

Technical Field

[0001] The invention belongs to the technical field of plant RNA extraction, and particularly relates to a kit and a method for extracting plant RNA based on magnetic beads. Background Art

[0002] In plant biology research, RNA is the key to understanding gene expression patterns, regulatory mechanisms, and their responses to environmental changes. With the rapid development of molecular biology and genomics, the demand for high-quality RNA is increasing. This demand not only covers conventional gene expression analysis, but also extends to advanced applications such as transcriptome sequencing, small RNA research, and gene function annotation. However, compared with animal tissue cells, plant cells are more complex, especially polysaccharide and polyphenol plants. Compared with ordinary plants, the content of secondary metabolites such as polysaccharides and polyphenols is higher, which makes it more difficult to separate and extract RNA. Among them, the polysaccharide component easily causes the viscosity of the solution of polysaccharide and polyphenol plant samples after treatment with lysis buffer, and DNA and RNA bind to polysaccharides during separation. When the washing solution cannot handle the polysaccharide component, it will affect the quality of DNA digestion and RNA elution, and ultimately lead to low RNA extraction purity. On the other hand, polyphenol components are extremely easy to oxidize, and the quinones formed by oxidation undergo aromatic ring addition with DNA / RNA to form an irreversible stable combination, resulting in low RNA extraction purity.

[0003] Common plant RNA extraction methods are Trizol methods, which involve toxic and harmful substances such as phenol and chloroform, pose a great threat to the safety of experimenters, and the method requires manual operation throughout the process, cannot be applied to large-scale extraction, and cannot extract RNA for polysaccharide polyphenol type plants. While another safer magnetic bead method avoids toxic and harmful components, such as the simplified extraction method of polyphenol plant RNA based on nanomagnetic beads disclosed in Chinese patent document CN107523565A, the RNA extracted by the method also includes other impurity fragments. Commercially available magnetic bead RNA extraction kits such as Tiangen brand and Promega brand, the polysaccharide polyphenol plant species extracted are relatively simple, and the plant sample RNA extraction with particularly high polysaccharide and polyphenol content cannot be satisfied, such as old leaves of cotton, old leaves of loquat, old leaves of agarwood, etc.

[0004] Therefore, it is necessary to provide a new kit for extracting plant RNA based on magnetic beads to solve the shortcomings of existing kits. Summary of the invention

[0005] The object of the present invention is to provide a kit and method for extracting plant RNA based on magnetic beads. In the present invention, by optimizing the components in the lysate, the DNA / RNA in the plant sample is fully separated from the impurity components, thereby extracting plant RNA with higher purity and concentration.

[0006] In a first aspect, the present invention provides a kit for extracting plant RNA based on magnetic beads, comprising a lysate, a binding solution, a magnetic bead suspension, a first washing solution, a DNaseⅠ reaction solution, a second washing solution and an elution solution; wherein the lysate comprises the following components: 10-300mM sodium tetraborate, 1-200mM EDTA, 0.5-15% by mass of hexadecyltrimethylammonium bromide, 0.5-15% by mass of sodium dodecyl sulfate, 0.5-15% by mass of sodium dodecyl sulfonate, 1-10% by mass of Triton X-100, 1-10% by mass of Tween 80, 0.1-3M sodium chloride, and 10-300mM of tris(hydroxymethyl)aminomethane.

[0007] In the present invention, the inventors have found that by adding sodium tetraborate, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, sodium dodecyl sulfonate, and surfactants Triton X-100 and Tween 80 to the lysis solution, the above components work synergistically to fully separate the DNA / RNA from the impurity components (polysaccharides, polyphenols) in the plant sample, thereby extracting plant RNA with higher purity and concentration.

[0008] In some preferred embodiments, the lysate includes the following components: 10-200 mM sodium tetraborate, 10-200 mM EDTA, 0.5-5% by mass of hexadecyltrimethylammonium bromide, 0.5-5% by mass of sodium dodecyl sulfate, 0.5-5% by mass of sodium dodecyl sulfonate, 5-10% by mass of Triton X-100, 5-10% by mass of Tween 80, 0.1-2 M sodium chloride, and 10-200 mM tris(hydroxymethyl)aminomethane.

[0009] In some more preferred embodiments, the lysate includes the following components: 150 mM sodium tetraborate, 100 mM EDTA, 5% by mass of hexadecyltrimethylammonium bromide, 5% by mass of sodium dodecyl sulfate, 5% by mass of sodium dodecyl sulfonate, 5% by mass of Triton X-100, 5% by mass of Tween 80, 2 M sodium chloride, and 150 mM tris(hydroxymethyl)aminomethane.

[0010] In some embodiments, the binding solution includes the following components: 40-90% by mass of isopropanol, 10-300 mM of sodium acetate, 10-300 mM of potassium acetate, and 1-10% by mass of polyvinyl pyrrolidone; wherein the polyvinyl pyrrolidone includes at least one of polyvinyl pyrrolidone K23-27 and polyvinyl pyrrolidone K88-96.

[0011] In the present invention, the inventors further discovered that adding sodium acetate, potassium acetate and polyvinyl pyrrolidone of a specific molecular weight to the binding solution can effectively prevent the oxidation products of the impurity components (polyphenols) in the plant samples from binding to DNA / RNA, thereby resulting in lower purity and concentration of the extracted RNA.

[0012] In some preferred embodiments, the binding solution includes the following components: 50-80% by mass of isopropanol, 50-200 mM of sodium acetate, 50-200 mM of potassium acetate, and 1-5% by mass of polyvinyl pyrrolidone.

[0013] In some more preferred embodiments, the binding solution includes the following components: 70% by mass of isopropanol, 150 mM of sodium acetate, 150 mM of potassium acetate, and 5% by mass of polyvinyl pyrrolidone.

[0014] In some embodiments, the concentration of the magnetic beads in the magnetic bead suspension is 0.5-5 mg / mL, the magnetic beads are silica-based magnetic beads with hydroxyl and / or carboxyl groups modified on the surface, and the particle size of the magnetic beads is 10-600 nm.

[0015] In some preferred embodiments, the concentration of the magnetic beads in the magnetic bead suspension is 0.5-4 mg / mL, the magnetic beads are silicon-based magnetic beads with hydroxyl groups modified on the surface, and the particle size of the magnetic beads is 20-500 nm.

[0016] In some more preferred embodiments, the particle size of the magnetic beads is 20-400 nm, and even more preferably, the particle size of the magnetic beads is 50-300 nm.

[0017] In some embodiments, the first washing solution includes the following components: 1-6M guanidine thiocyanate, 1-50mM EDTA, 40-80% by mass ethanol, 0.5-10% by mass NP-40, 0.5-10% by mass hexadecyltrimethylammonium bromide, and 0.5-10% by mass sodium dodecyl sulfate.

[0018] In some embodiments, the DNase I reaction solution contains 5-20 U of DNase I; the second washing solution includes 55-85% by mass ethanol; and the eluent includes DEPC water.

[0019] It is understandable that the DNase I reaction solution can be routinely selected according to actual use needs, for example, the DNase I reaction solutions of Abotek, Zhuhai Baorui, Novezan, Promega, NEB and Takara can be used.

[0020] In some preferred embodiments, the preparation of DEPC water comprises: dissolving 1-2 mL of DEPC (Diethylpyrocarbonate) in 1 L of pure water, stirring overnight and sterilizing under high pressure at 121° C. for 20 min, and cooling to obtain DEPC water.

[0021] In some embodiments, the plant is a plant rich in polysaccharides and / or polyphenols, and the plant includes at least one of cotton, loquat, and agarwood.

[0022] It can be understood that when the kit provided in the present invention is used to extract plant RNA, the plant can be a conventional plant in the prior art, for example, a plant rich in polysaccharides and / or polyphenols, and can also preferably include at least one of cotton, loquat, and agarwood; and the plant can be selected from at least one of plant leaves, roots, and stems.

[0023] In a second aspect, the present invention provides a method for extracting plant RNA using any of the above-mentioned kits, comprising the following steps: S1, mixing the plant sample to be extracted with a lysate, oscillating and mixing, incubating at room temperature, and centrifuging to obtain a supernatant; S2, adding a binding solution and a magnetic bead suspension to the supernatant, binding at room temperature, and separating to obtain a nucleic acid-magnetic bead complex; S3, using a first washing solution, a DNaseⅠ reaction solution, and a second washing solution to treat the nucleic acid-magnetic bead complex, respectively, to obtain a purified nucleic acid-magnetic bead complex; S4, using an elution solution to elute the purified nucleic acid-magnetic bead complex to obtain plant RNA.

[0024] The extraction method provided by the invention is simple and efficient, and the kit can be used to extract plant RNA manually or by using an automated nucleic acid extraction device.

[0025] In some embodiments, the mass volume ratio of the plant sample to be extracted and the lysate is (100-300 mg): (250-600 μL); the oscillation mixing time is 10-60 s, and the room temperature incubation time is 1-30 min; the centrifugation treatment specifically includes: centrifugation for 5-10 min at 0-4°C and a rotation speed of 10000-12000 rpm.

[0026] In some embodiments, in step S1, before mixing the plant sample to be extracted with the lysis solution, a step of pretreating the plant sample to be extracted is also included; wherein the pretreatment step specifically includes: grinding the plant sample to be extracted with liquid nitrogen to obtain a pretreated plant sample to be extracted; wherein the liquid nitrogen grinding time is 1-5 minutes.

[0027] In some embodiments, in step S2, the amount of binding solution added is 300-500 μL, and the amount of magnetic bead suspension added is 300-800 μL; in step S3, the amount of the first washing solution is 300-800 μL, the amount of DNase I reaction solution is 30-200 μL, and the amount of the second washing solution is 500-1000 μL; in step S4, the amount of elution solution is 30-200 μL.

[0028] The beneficial effects of the present invention are as follows: different from the prior art, the present invention adds sodium tetraborate, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, sodium dodecyl sulfonate, surfactant Triton X-100, and Tween 80 into the lysis solution of the kit, and the above components act synergistically to fully separate DNA / RNA from impurity components (polysaccharides, polyphenols) in the plant sample, thereby extracting plant RNA with higher purity, concentration and better integrity; therefore, the kit has good application prospects in the extraction of plant RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of agarose electrophoresis of RNA extracted from old cotton leaves in Example 1 and Comparative Example 1;

[0030] Figure 2 The results of agarose electrophoresis of RNA extracted from old leaves of Aquilaria sinensis in Example 4 and Comparative Example 2;

[0031] Figure 3 The elution product results of RNA extracted from old cotton leaves in Example 1 and Comparative Example 3;

[0032] Figure 4 The elution product results of RNA extracted from old cotton leaves in Example 1 and Comparative Example 4;

[0033] Figure 5 The results of agarose electrophoresis of RNA extracted from old cotton leaves in Example 1 and Comparative Example 4;

[0034] Figure 6 The elution product results of RNA extracted from old cotton leaves in Example 1 and Comparative Example 5;

[0035] Figure 7 The results of agarose electrophoresis of RNA extracted from old cotton leaves in Example 1 and Comparative Example 5;

[0036] Figure 8 The elution product results of RNA extracted from old cotton leaves in Example 1 and Comparative Example 6;

[0037] Fig. 9 These are the agarose electrophoresis results of RNA extracted from old cotton leaves in Example 1 and Comparative Example 6. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0040] Example 1

[0041] In this embodiment, the kit for extracting plant RNA based on magnetic beads includes a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a DNase I reaction solution, a second washing solution and an elution solution;

[0042] The lysate comprises the following components: 150 mM sodium tetraborate, 100 mM EDTA, 5% by mass of hexadecyltrimethylammonium bromide, 5% by mass of sodium dodecyl sulfate, 5% by mass of sodium dodecyl sulfonate, 5% by mass of Triton X-100, 5% by mass of Tween 80, 2 M sodium chloride, and 150 mM tris(hydroxymethyl)aminomethane;

[0043] The binding solution includes the following components: 70% by mass of isopropanol, 150 mM of sodium acetate, 150 mM of potassium acetate, and 5% by mass of polyvinyl pyrrolidone K23-27;

[0044] The concentration of magnetic beads in the magnetic bead suspension is 3 mg / mL, the magnetic beads are silicon-based magnetic beads with hydroxyl groups modified on the surface, and the particle size of the magnetic beads is 20-400 nm, and the concentration of alcohol contained in the suspension is 50-80%;

[0045] The first washing solution comprises the following components: 3M guanidine thiocyanate, 25mM EDTA, 60% by mass ethanol, 5% by mass NP-40, 5% by mass cetyltrimethylammonium bromide, and 5% by mass sodium dodecyl sulfate;

[0046] The DNaseⅠ reaction solution contained 20U of DNaseⅠ (Zhuhai Baorui);

[0047] The second washing liquid comprises 70% by mass ethanol;

[0048] The eluent included DEPC water.

[0049] The kit in this example was used to extract total RNA from old cotton leaves using a NanoMagBio S-48 fully automatic nucleic acid extractor, and three old cotton leaf samples were used for repeated testing, wherein the instrument parameter settings are shown in Table 1 below.

[0050] Table 1 Procedure for extracting total RNA from plants using the NanoMagBio S-48 fully automatic nucleic acid extractor

[0051] step 1 step 2 Steps 3 Steps 4 Steps 5 Steps Step 6 Step 7 Step 8 Step 9 Workstation 4 1 2 3 4 6 6 5 4 Waiting time / / / / / / / 3min / Blending Mode 1 1 3 3 3 1 1 3 3 Mixing time 30s 5min 2min 2min 2min 15min 5min 4min 30s Suspend no no no no no yes no no no Magnetic attraction time 1min 2min 1min 1min 1min / 1min 1min / temperature / 25℃ / / / / / 65℃ /

[0052] Take out a 96-well plate and add the components of the kit into the corresponding wells of the 96-well plate in sequence according to Table 2 below.

[0053] Table 2 The amount of each component reagent in the kit and the corresponding position of the 96-well plate

[0054]

[0055]

[0056] The specific steps for RNA extraction are as follows:

[0057] 1) Take the plant samples frozen at -80℃, grind them with liquid nitrogen, and then take 2mL centrifuge tubes to pack the ground sample powder. Each centrifuge tube should be filled with 200mg of plant sample powder. After packing, keep the temperature low in liquid nitrogen environment;

[0058] 2) Add 500 μL of lysis solution to the centrifuge tube containing the plant sample powder, and quickly mix on a vortex mixer for 30 seconds, then incubate at room temperature for 15 minutes. After incubation, centrifuge at 4°C and 11,000 rpm for 8 minutes;

[0059] 3) Transfer 450 μL of the supernatant after the above centrifugation to the 1 / 7 column of the 96-well plate, and place the 96-well plate into the card slot of the instrument, place the magnetic rod cover, close the door, and start running the program;

[0060] 4) After the program is paused, take out the 96-well plate, add 800 μL of the second washing solution to the 6 / 12 column, then put the 96-well plate back into the instrument's card slot and continue running the program;

[0061] 5) After the program is completed, take out the 96-well plate and transfer the 5 / 11 columns of samples to a clean RNase-free centrifuge tube, which is the extracted cotton old leaf RNA.

[0062] Example 2

[0063] In this embodiment, the kit for extracting plant RNA based on magnetic beads includes a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a DNase I reaction solution, a second washing solution and an elution solution;

[0064] The lysate comprises the following components: 10 mM sodium tetraborate, 200 mM EDTA, 15% by mass of hexadecyltrimethylammonium bromide, 15% by mass of sodium dodecyl sulfate, 0.5% by mass of sodium dodecyl sulfonate, 10% by mass of Triton X-100, 1% by mass of Tween 80, 3 M sodium chloride, and 10 mM tris(hydroxymethyl)aminomethane;

[0065] The binding solution includes the following components: 40% by mass of isopropanol, 300 mM of sodium acetate, 10 mM of potassium acetate, and 10% by mass of polyvinyl pyrrolidone K23-27;

[0066] The concentration of magnetic beads in the magnetic bead suspension is 3 mg / mL, the magnetic beads are silicon-based magnetic beads with hydroxyl groups modified on the surface, and the particle size of the magnetic beads is 20-400 nm, and the concentration of alcohol contained in the suspension is 50-80%;

[0067] The first washing solution comprises the following components: 1M guanidine thiocyanate, 50mM EDTA, 80% by mass ethanol, 10% by mass NP-40, 10% by mass hexadecyltrimethylammonium bromide, and 0.5% by mass sodium dodecyl sulfate;

[0068] The DNaseⅠ reaction solution contained 20U of DNaseⅠ (Zhuhai Baorui);

[0069] The second washing liquid comprises 70% by mass ethanol;

[0070] The eluent included DEPC water.

[0071] The method of extracting total RNA from old cotton leaves using the kit in this example and NanoMagBio S-48 fully automatic nucleic acid extractor is the same as that in Example 1, and RNA from old cotton leaves is extracted.

[0072] Example 3

[0073] In this embodiment, the kit for extracting plant RNA based on magnetic beads includes a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a DNase I reaction solution, a second washing solution and an elution solution;

[0074] The lysate comprises the following components: 200 mM sodium tetraborate, 10 mM EDTA, 0.5% by mass of hexadecyltrimethylammonium bromide, 0.5% by mass of sodium dodecyl sulfate, 15% by mass of sodium dodecyl sulfonate, 1% by mass of Triton X-100, 10% by mass of Tween 80, 0.1 M sodium chloride, and 300 mM tris(hydroxymethyl)aminomethane;

[0075] The binding solution includes the following components: 90% by mass of isopropanol, 10 mM of sodium acetate, 300 mM of potassium acetate, and 1% by mass of polyvinyl pyrrolidone K23-27;

[0076] The concentration of magnetic beads in the magnetic bead suspension is 3 mg / mL, the magnetic beads are silicon-based magnetic beads with hydroxyl groups modified on the surface, and the particle size of the magnetic beads is 20-400 nm, and the concentration of alcohol contained in the suspension is 50-80%;

[0077] The first washing solution comprises the following components: 6M guanidine thiocyanate, 1mM EDTA, 40% by mass ethanol, 0.5% by mass NP-40, 0.5% by mass hexadecyltrimethylammonium bromide, and 10% by mass sodium dodecyl sulfate;

[0078] The DNaseⅠ reaction solution contained 20U of DNaseⅠ (Zhuhai Baorui);

[0079] The second washing liquid comprises 70% by mass ethanol;

[0080] The eluent included DEPC water.

[0081] The method of extracting total RNA from old cotton leaves using the kit in this example and NanoMagBio S-48 fully automatic nucleic acid extractor is the same as that in Example 1, and RNA from old cotton leaves is extracted.

[0082] Example 4

[0083] In this example, the components in the kit for extracting plant RNA based on magnetic beads are basically the same as those in Example 1, except that polyvinylpyrrolidone K23-27 is replaced by polyvinylpyrrolidone K88-96 in the binding solution.

[0084] The total RNA from the old leaves of Aquilaria sinensis was extracted using the kit in this example using the NanoMagBio S-48 fully automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and the RNA from the old leaves of Aquilaria sinensis was extracted.

[0085] Comparative Example 1

[0086] In this comparative example, the commercially available Tiangen brand polysaccharide and polyphenol plant RNA kit was used to extract total RNA from the old cotton leaves in Example 1, and three old cotton leaf samples were used for repeated testing, and the extraction operation was performed according to the instructions of the kit of this brand; RNA from the old cotton leaves was extracted.

[0087] Comparative Example 2

[0088] In this comparative example, a commercially available Huayueyang brand polysaccharide and polyphenol plant RNA kit was used to extract the total RNA of the old leaves of Aquilaria sinensis in Example 4, and three samples of old leaves of Aquilaria sinensis were used for repeated testing, and the extraction operation was carried out according to the instructions of the kit of this brand; RNA of the old leaves of Aquilaria sinensis was extracted.

[0089] Comparative Example 3

[0090] In this comparative example, the components in the kit for extracting plant RNA based on magnetic beads are basically the same as those in Example 1, except that sodium tetraborate and Tween 80 are not added to the lysis solution.

[0091] The kit in this example was used to extract the total RNA from the old cotton leaves in Example 1 using a NanoMagBio S-48 fully automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and RNA from the old cotton leaves was extracted.

[0092] Comparative Example 4

[0093] In this comparative example, the components in the kit for extracting plant RNA based on magnetic beads are basically the same as those in Example 1, except that polyvinyl pyrrolidone K23-27 is not added to the binding solution.

[0094] The kit in this example was used to extract the total RNA from the old cotton leaves in Example 1 using a NanoMagBio S-48 fully automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and RNA from the old cotton leaves was extracted.

[0095] Comparative Example 5

[0096] In this comparative example, the components in the kit for extracting plant RNA based on magnetic beads are basically the same as those in Example 1, except that polyvinyl pyrrolidone K88-96 is added to the binding solution.

[0097] The kit in this example was used to extract the total RNA from the old cotton leaves in Example 1 using a NanoMagBio S-48 fully automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and RNA from the old cotton leaves was extracted.

[0098] Comparative Example 6

[0099] In this comparative example, the components in the kit for extracting plant RNA based on magnetic beads are basically the same as those in Example 1, except that polyvinyl pyrrolidone K13-18 is added to the binding solution.

[0100] The kit in this example was used to extract the total RNA from the old cotton leaves in Example 1 using a NanoMagBio S-48 fully automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and RNA from the old cotton leaves was extracted.

[0101] Performance Testing

[0102] The cotton old leaf RNA extracted in Examples 1-3 and Comparative Examples 1 and 3-6, and the agarwood old leaf RNA extracted in Example 4 and Comparative Example 2 were subjected to quality testing. Specifically, the RNA A was detected using a spectrophotometer. 230nm , A 260nm and A 280nm The absorbance values ​​at are shown in Table 1 below.

[0103] Table 1 Test results

[0104]

[0105]

[0106] In the table, “ / ” indicates that RNA could not be extracted.

[0107] As can be seen from Table 1, the average RNA concentration of cotton old leaves extracted using the kit in Example 1 was 291.427 ng / μL, the elution volume was about 100 μL, the total yield was about 30 μg, and A 260nm / A 280nm The ratios are all above 2.0, A 260nm / A 230nm The ratio is about 2.2; the average RNA concentration of cotton old leaves extracted using the kit in Example 2 is 283.345 ng / μL, the elution volume is about 100 μL, the total yield is about 28 μg, A 260nm / A 280nm The ratios are all above 2.0, A 260nm / A 230nm The ratio is about 2.1; the average RNA concentration of cotton old leaves extracted using the kit in Example 3 is 286.122 ng / μL, the elution volume is about 100 μL, the total yield is about 29 μg, A 260nm / A 280nm The ratios are all above 2.0, A 260nm / A 230nmThe ratio is about 2.1; the average RNA concentration of the old leaves of Aquilaria sinensis extracted using the kit in Example 4 is 190.485 ng / μL, the elution volume is about 100 μL, the total yield is about 20 μg, A 260nm / A 280nm The ratios are all above 2.0, A 260nm / A 230nm The ratio is about 2.0; the above results show that the kit of the present invention can be used to extract plant RNA with high purity and concentration.

[0108] In Comparative Example 1, the commercially available Tiangen brand polysaccharide and polyphenol plant RNA kit was used to extract RNA from old cotton leaves. The results showed that the average concentration of RNA extracted from old cotton leaves was 237.268 ng / μL, the elution volume was about 100 μL, the total yield was about 23 μg, and A 260nm / A 280nm The ratio is about 2.0, A 260nm / A 230nm The ratio is less than 1; the purity and concentration of the RNA extracted from old cotton leaves in this comparative example are significantly lower than those in Example 1.

[0109] In Comparative Example 2, the commercially available Huayueyang brand polysaccharide polyphenol plant RNA kit was used to extract RNA from old leaves of Aquilaria sinensis. The results showed that the average concentration of RNA extracted from old leaves of Aquilaria sinensis was 336.968 ng / μL, the elution volume was about 100 μL, the total yield was about 34 μg, and A 260nm / A 280nm The ratio is about 1.3, A 260nm / A 230nm The ratio is less than 1; the purity of the RNA extracted from old cotton leaves in this comparative example is significantly lower than that in Example 4.

[0110] Comparative Example 3 Compared with Example 1, sodium tetraborate and Tween 80 were not added to the lysate. The results showed that the extracted eluate was in a relatively viscous state, and the magnetic beads and the eluate were inseparable. Therefore, pure RNA was not successfully extracted, and the RNA concentration and A could not be determined. 260nm / A 280nm Ratio and A 260nm / A 230nm ratio.

[0111] Comparative Example 4 Compared with Example 1, polyvinyl pyrrolidone K23-27 was not added to the binding solution. The results showed that the average RNA concentration of the extracted cotton old leaves was 438.618 ng / μL, the elution volume was about 100 μL, the total yield was about 43 μg, and A 260nm / A 280nm The ratio is about 1, A 260nm / A 230nmThe ratio is less than 1; the purity of the RNA extracted from old cotton leaves in this comparative example is significantly lower than that in Example 1.

[0112] Comparative Example 5 Compared with Example 1, polyvinyl pyrrolidone K88-96 was added to the binding solution. The results showed that the average RNA concentration of the extracted cotton old leaves was 628.675 ng / μL, the elution volume was about 100 μL, the total yield was about 63 μg, and A 260nm / A 280nm The ratio is about 1, A 260nm / A 230nm The ratio is less than 1; the purity of the RNA extracted from old cotton leaves in this comparative example is significantly lower than that in Example 1.

[0113] Comparative Example 6 Compared with Example 1, polyvinyl pyrrolidone K13-18 was added to the binding solution. The results showed that the average RNA concentration of the extracted cotton old leaves was 565.415 ng / μL, the elution volume was about 100 μL, the total yield was about 56 μg, and A 260nm / A 280nm The ratio is about 1.4, A 260nm / A 230nm The ratio is less than 1; the purity of the RNA extracted from old cotton leaves in this comparative example is significantly lower than that in Example 1.

[0114] The above results show that in the present invention, sodium tetraborate, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, sodium dodecyl sulfonate, surfactants Triton X-100, and Tween 80 are added to the lysate, and the above components act synergistically to fully separate the DNA / RNA from the impurity components (polysaccharides, polyphenols) in the plant sample, and the addition of sodium acetate, potassium acetate and polyvinyl pyrrolidone of a specific molecular weight to the binding solution can effectively prevent the oxidation products of the impurity components (polyphenols) in the plant sample from binding to the DNA / RNA, and finally extract the plant RNA with higher purity and concentration.

[0115] Further, 1.2% ordinary agarose gel electrophoresis was performed on the RNA of old cotton leaves extracted in Example 1 and Comparative Example 1, and the RNA of old agarwood leaves extracted in Example 4 and Comparative Example 2, respectively, and the state of the eluate of the RNA of old cotton leaves extracted in Example 1 and Comparative Example 3 was observed, as well as the state of the eluate of the RNA of old cotton leaves extracted in Example 1 and Comparative Example 4, the state of the eluate of the RNA of old cotton leaves extracted in Example 1 and Comparative Example 5, and the state of the eluate of the RNA of old cotton leaves extracted in Example 1 and Comparative Example 6 were observed and subjected to 1.2% ordinary agarose gel electrophoresis to evaluate the integrity of the extracted RNA, and the results were as follows: Figure 1-9 shown.

[0116] The principle of RNA integrity evaluation is as follows: 28S and 18S are the two main subunits of eukaryotic rRNA (ribosomal RNA). Various pathways of degradation may occur during RNA extraction. 28S / 18S is an indicator to measure the integrity of the extracted RNA. If 28S / 18S is 1.8-2.0, it indicates that the extracted RNA has good integrity and basically no degradation occurs. The electrophoresis band should be 28S on the top and 18S on the bottom, and the brightness of 28S is twice that of 18S. It is necessary to use image analysis tools to judge the brightness ratio of the bands, but it can usually be observed by the naked eye. The 28S band is obviously brighter than the 18S band, and the band is not diffuse, which indicates that the extracted RNA has good integrity.

[0117] from Figure 1 It can be seen that the 28S and 18S bands of the RNA extracted by the kit in Comparative Example 1 are significantly darker than those in Example 1, and the electrophoresis bands are more diffuse.

[0118] from Figure 2 It can be seen that the RNA extracted by the kit in Comparative Example 2 has no obvious 28S and 18S bands, and the bright electrophoresis spot wells in Comparative Example 2 indicate that there are residual impurities such as protein and polysaccharide. The 28S and 18S bands of the RNA extracted by the kit in Example 4 are clear without obvious tailing and diffusion, indicating that the extracted RNA has good integrity and high quality.

[0119] from Figure 3 It can be seen that the RNA extracted by the kit in Comparative Example 3 is of poor quality, the eluent is viscous, the magnetic beads and the eluent cannot be separated, and there are more magnetic beads remaining in the eluent.

[0120] from Figure 4 It can be seen that the RNA extracted by the kit in Comparative Example 4 is of poor quality, the eluent is viscous, the magnetic beads and the eluent cannot be separated, and some magnetic beads remain in the eluent.

[0121] from Figure 5 It can be seen that the 28S and 18S bands of the RNA extracted by the kit in Comparative Example 4 are more diffuse than those in Example 1, and the bright electrophoresis spots indicate that impurities such as proteins and polysaccharides remain.

[0122] from Figure 6 It can be seen that the RNA extracted by the kit in Comparative Example 5 is of poor quality and the eluate is turbid.

[0123] from Figure 7 It can be seen that the 28S and 18S bands of the RNA extracted by the kit in Comparative Example 5 are not significantly different from those in Example 1.

[0124] from Figure 8 It can be seen that the RNA extracted by the kit in Comparative Example 6 is of poor quality and the eluate is turbid.

[0125] from Fig. 9 It can be seen that the 28S and 18S bands of the RNA extracted by the kit in Comparative Example 6 are not significantly different from those in Example 1.

[0126] The above results show that plant RNA with better integrity can be extracted by using the kit of the present invention.

[0127] In summary, the kit of the present invention can be used to extract plant RNA with better purity, concentration and integrity.

[0128] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0129] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A kit for extracting plant RNA based on magnetic beads, characterized in that: It includes a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a DNaseⅠ reaction solution, a second washing solution and an elution solution; The lysate comprises the following components: 10-300 mM sodium tetraborate, 1-200 mM EDTA, 0.5-15% by mass of hexadecyltrimethylammonium bromide, 0.5-15% by mass of sodium dodecyl sulfate, 0.5-15% by mass of sodium dodecyl sulfonate, 1-10% by mass of Triton X-100, 1-10% by mass of Tween 80, 0.1-3 M sodium chloride, and 10-300 mM of tris(hydroxymethyl)aminomethane.

2. The kit for extracting plant RNA based on magnetic beads according to claim 1, characterized in that The binding solution comprises the following components: 40-90% by mass of isopropanol, 10-300 mM of sodium acetate, 10-300 mM of potassium acetate, and 1-10% by mass of polyvinyl pyrrolidone; Wherein, the polyvinyl pyrrolidone includes at least one of polyvinyl pyrrolidone K23-27 and polyvinyl pyrrolidone K88-96.

3. The kit for extracting plant RNA based on magnetic beads according to claim 1, characterized in that The concentration of the magnetic beads in the magnetic bead suspension is 0.5-5 mg / mL, the magnetic beads are silicon-based magnetic beads with hydroxyl and / or carboxyl groups modified on the surface, and the particle size of the magnetic beads is 10-600 nm.

4. The kit for extracting plant RNA based on magnetic beads according to claim 1, characterized in that The first washing solution comprises the following components: 1-6M guanidine thiocyanate, 1-50mM EDTA, 40-80% by mass ethanol, 0.5-10% by mass NP-40, 0.5-10% by mass hexadecyltrimethylammonium bromide, and 0.5-10% by mass sodium dodecyl sulfate.

5. The kit for extracting plant RNA based on magnetic beads according to claim 1, characterized in that: The DNaseⅠ reaction solution contains 5-20U DNaseⅠ; The second washing liquid comprises 55-85% by mass of ethanol; The eluent includes DEPC water.

6. The kit for extracting plant RNA based on magnetic beads according to claim 1, characterized in that: The plant is a plant rich in polysaccharides and / or polyphenols, and the plant includes at least one of cotton, loquat, and agarwood.

7. A method for extracting plant RNA using the kit according to any one of claims 1 to 6, characterized in that: The steps include: S1. Mix the plant sample to be extracted with the lysis solution, shake and mix, incubate at room temperature, and then centrifuge to obtain a supernatant; S2, adding a binding solution and a magnetic bead suspension to the supernatant, and after binding at room temperature, performing separation treatment to obtain a nucleic acid-magnetic bead complex; S3, using the first washing solution, the DNase I reaction solution, and the second washing solution to treat the nucleic acid-magnetic bead complex respectively to obtain a purified nucleic acid-magnetic bead complex; S4. Using an elution solution to elute the purified nucleic acid-magnetic bead complex to obtain plant RNA.

8. The method according to claim 7, characterized in that In step S1, the mass volume ratio of the plant sample to be extracted and the lysate is (100-300 mg): (250-600 μL); The oscillation mixing time is 10-60s, and the incubation time at room temperature is 1-30min; The centrifugation treatment specifically includes: centrifuging for 5-10 minutes at 0-4°C and a rotation speed of 10000-12000 rpm.

9. The method according to claim 7, characterized in that: In step S1, before mixing the plant sample to be extracted with the lysis solution, the method further includes a step of pre-treating the plant sample to be extracted; The pretreatment step specifically includes: grinding the plant sample to be extracted with liquid nitrogen to obtain a pretreated plant sample to be extracted; The liquid nitrogen grinding time is 1-5 minutes.

10. The method according to claim 7, characterized in that In step S2, the amount of the binding solution added is 300-500 μL, and the amount of the magnetic bead suspension added is 300-800 μL; In step S3, the amount of the first washing solution is 300-800 μL, the amount of the DNase I reaction solution is 30-200 μL, and the amount of the second washing solution is 500-1000 μL; In step S4, the amount of the eluent used is 30-200 μL.

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