Plant sample RNA extraction kit and extraction method
By providing a plant RNA extraction kit containing specific reagents and steps, the problems of RNA easy to degrade and interfering substances in the cell during plant RNA extraction are solved, and the stable extraction and high yield of high-quality RNA is achieved, which is suitable for molecular biology research.
Patent Information
- Application Number
- CN202510184566.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-25
AI Technical Summary
It is difficult to obtain high-quality plant RNA in the prior art, mainly due to the ease of RNA degradability, the presence of RNA enzymes, and the interference of polysaccharides, phenols and other secondary metabolites in plant cells, resulting in low RNA yield, unstable extraction effect, low purity and poor quality.
A plant sample RNA extraction kit is provided, including abrasive material, lysate, isopropanol, nucleic acid adsorption column, rinse solution I, rinse solution II and eluent. CTAB and 2-mercaptoethanol were added to the lysate, and stable RNA extraction was achieved through grinding and centrifugation steps combined with the use of a nucleic acid adsorption column.
This method can effectively remove polysaccharides, phenols and RNase, improve the integrity and yield of RNA, obtain high-quality plant RNA, and have stable extraction effect, and is suitable for further molecular biology research.
Smart Images
Figure CN119876122A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a plant sample RNA extraction kit and an extraction method. Background Art
[0002] Plant RNA plays an extremely important role in plant life activities. RNA is a key molecule in plant gene expression regulation and is involved in a variety of life processes, including growth and development, gene expression regulation, and stress resistance. By extracting total RNA from plants, we can study the expression pattern and regulatory mechanism of genes and reveal the function and regulatory mechanism of genes. High-quality RNA is the basis and prerequisite for subsequent molecular biology research such as gene cloning and transformation, cDNA library construction, and RNA-Seq.
[0003] However, RNA is more easily degraded than DNA, and the presence of RNAse (RNase) can cause RNA to be decomposed. In addition, plant cells contain a large amount of polysaccharides, phenols, and other secondary metabolites, which are easily mixed with RNA during the extraction process, affecting the extraction effect. Plant tissues contain high levels of RNase, high water content, and difficult-to-decompose fibrous tissues (such as lignin) all increase the difficulty of extraction. In conventional methods, only some polysaccharides can be removed through SDS-guanidine hydrochloride or Licl precipitation in a high-concentration salt ion environment, and high-quality plant RNA cannot be obtained, resulting in low plant RNA yield, unstable extraction effect, low purity, poor quality, and difficulty in advancing further research. Summary of the invention
[0004] In order to solve at least one of the above problems, the present invention provides a plant sample RNA extraction kit and an extraction method.
[0005] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a plant sample RNA extraction kit, comprising a grinding material, a lysis solution, isopropanol, a nucleic acid adsorption column, a rinsing solution I, a rinsing solution II, and an eluent; The grinding material is a 3-6 mm steel ball; The lysis buffer includes pH 9.0, 80-100 mM Tris, 120-150 mM NaCl, 30-50 mM EDTA, 1.2-1.5% CTAB and 1.2-1.5% 2-mercaptoethanol; The rinse solution I comprises 500 g / L guanidine hydrochloride, 50% by volume ethanol, and 16% by volume nuclease-free water; The rinse solution II includes 50 mM Tris-HCl, 80% ethanol by volume, and 17.5% nuclease-free water by volume; The elution solution is nuclease-free water.
[0006] In some embodiments of the present invention, the grinding material includes 4 mm steel balls and 6 mm steel balls.
[0007] In some embodiments of the present invention, the nucleic acid adsorption column is an RNA extraction column.
[0008] The second aspect of the present invention provides a method for stably extracting RNA from a plant sample using the kit described in the first aspect, comprising the following steps: S1. Weigh the plant sample to be extracted, grind the material and place it in sample tube A, and pre-cool it in liquid nitrogen; S2. Add lysis buffer to pre-cooled sample tube A and shake to mix. Grind in a grinder at 4°C and 40 Hz for 20-30 seconds. After grinding thoroughly, centrifuge at 4°C and 10,000-12,000 rpm for 8-10 minutes, and transfer the supernatant to sample tube B. S3. Add isopropanol of the same volume as the supernatant solution to sample tube B and oscillate to mix; add the mixed solution in sample tube B to sample tube C assembled with a nucleic acid adsorption column, centrifuge at room temperature 10000-12000 rpm for 30 s, discard the filtrate, and reassemble the adsorption column back into sample tube C; S4, add rinse solution I to the sample tube C assembled with the nucleic acid adsorption column, centrifuge at room temperature, 10000-12000 rpm for 30 s, discard the filtrate, and reassemble the nucleic acid adsorption column back into the sample tube C; S5. Add 400 μL of washing solution II to the sample tube C assembled with the nucleic acid adsorption column, centrifuge at room temperature, 10000-12000 rpm for 30 s, and discard the filtrate; S6. Transfer the nucleic acid adsorption column to sample tube D, add 50-70 μL of nuclease-free water to the middle part of the adsorption membrane of the nucleic acid adsorption column, let it stand at room temperature for 2 min, centrifuge at 10000-12000 rpm at room temperature for 2 min, discard the nucleic acid adsorption column, and obtain the RNA solution.
[0009] In some embodiments of the present invention, in the grinding step of step S2, after grinding once, the sample tube can be taken out and placed on dry ice to check the grinding condition of the sample. If the grinding is not sufficient, grinding materials with different particle sizes can be added for grinding again.
[0010] In some embodiments of the present invention, after completing step S5, the nucleic acid adsorption column is reassembled into the sample tube C and centrifuged at room temperature, 10,000-12,000 rpm for 2 min to remove excess liquid in the nucleic acid adsorption column.
[0011] In some embodiments of the present invention, the step S4 is repeated 1-2 times by adding rinsing solution I for rinsing.
[0012] In some embodiments of the present invention, the step S5 is repeated 1-2 times to add rinsing solution II for rinsing.
[0013] In some embodiments of the present invention, the sample tube A is a grinding sample tube, and the sample tubes B, C, and D are EP tubes. The bottom of the grinding sample tube is thicker and sharper than that of the EP tube. The EP tubes are 1.5 mL and 2.0 mL EP tubes, and the adapter tubes can be selected according to the amount of lysate added or the experimental system. Generally, the intermediate tube for plant sample nucleic acid extraction uses a 2.0 mL EP tube, and the final RNA storage uses a 1.5 mL EP tube.
[0014] In some embodiments of the present invention, the plant sample to be extracted includes roots, stems, leaves, and seeds of the plant.
[0015] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides an extraction kit and method for stably extracting RNA from plant samples, the reagents and consumables used are simple and easy to obtain, and no special reagents and consumables need to be prepared; the operation is simple, and no extra operation steps need to be added in the experiment; the application scheme has strong pertinence, and the lysis system in the application scheme adds 1.5% CTAB, so that the plant tissue is fully and completely lysed, and polysaccharides, phenolic compounds, RNase and other metabolites that interfere with RNA extraction are removed to a large extent, thereby reducing the impact on RNA experimental results. High-quality RNA with high yield and high integrity can be obtained, and the multiple extraction effects of sample RNA are stable. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The peak diagrams of three wheat seed samples extracted in Example 1 of the present invention are shown; Figure 2 The peak diagrams extracted from three wheat seed samples in Comparative Example 1 of the present invention are shown; Figure 3 The peak diagrams of three wheat seed samples extracted in Comparative Example 2 of the present invention are shown; Figure 4 The peak diagrams of three wheat root samples extracted in Example 2 of the present invention are shown; Figure 5 The peak diagrams extracted from three wheat root samples in Comparative Example 4 of the present invention are shown; Figure 6 The peak diagrams of the extraction of three wheat root samples in Comparative Example 5 of the present invention are shown. DETAILED DESCRIPTION
[0017] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
[0019] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.
[0020] Example 1 1. Preparation before the experiment Prepare in advance: liquid nitrogen box, dry refrigerator, tweezers, scissors, carving knife, alcohol spray bottle, dust-free paper towels, pipette, and tip box.
[0021] (1) Before the experiment, wipe the pipette and test bench with 75% alcohol.
[0022] (2) Prepare a plate according to the extraction quantity, which is used to place the EP tube and is the main operating platform.
[0023] (3) Prepare a 2.0 mL grinding tube and add a clean 6 mm large steel ball to the grinding tube.
[0024] (4) Prepare 1.5 mL and 2.0 mL EP tubes and select the appropriate tubes according to the amount of lysate added or the experimental system.
[0025] (5) Find the necessary reagents and place them where they will not interfere with the operation.
[0026] (6) Scissors and tweezers must be sterilized under UV light for 30 minutes and wiped with 75% alcohol before use.
[0027] (7) Prepare a liquid nitrogen box and dry ice box in advance. Sampling must be done on dry ice to prevent tissue degradation.
[0028] (8) Pre-cool the centrifuge to 4°C.
[0029] (9) Preparation of lysis buffer 1: 100 mM Tris (pH 9.0), 150 mM NaCl, 50 mM EDTA, 1.5% CTAB, and 1.5% 2-mercaptoethanol (2-mercaptoethanol was added before use).
[0030] 2. Wheat Seed RNA Extraction (1) Add a 6 mm steel ball to a 2 mL grinding tube and write numbers 001, 002, and 003 on the tube cap and side wall; (2) Weigh 50 mg, 40 mg, and 30 mg of wheat seeds respectively and put them into grinding tubes numbered 001, 002, and 003, and quickly put them into liquid nitrogen for temporary storage; (3) Insert the sample into the pre-cooled grinder adapter, making sure that the sample is balanced in the adapter, and install the adapter into the grinder. Tighten the adapter cover and set the grinder operating parameters: 60 Hz, 60 s, start the device, and grind once. (4) After the equipment is finished running, take out the sample tube and place it on dry ice to check the sample grinding and tube breakage; (5) Add 700 μL of lysis buffer, shake vigorously for 15 s, and grind using a cryo-grinder at 40 Hz for 20 s at 4°C. (6) Centrifuge at 4°C, 12,000 rpm for 10 min, transfer the supernatant to a new 2 mL centrifuge tube, add an equal volume of isopropanol to the tube, and shake vigorously for 15 s. (7) Take 700 μL of the mixed solution into the nucleic acid adsorption column, centrifuge at room temperature and 12000 rpm for 30 s, discard the filtrate, and reassemble the nucleic acid adsorption column; if there is a lot of water phase, repeat this step, discard the waste liquid in the collection tube, press the collection tube mouth on clean toilet paper once, and do not press the same position repeatedly to prevent waste liquid residue and cross contamination, and put the nucleic acid adsorption column back into the collection tube; (8) Add 400 μL of washing solution I to the nucleic acid adsorption column, centrifuge at room temperature, 12,000 rpm for 30 s, repeat this step once, discard the filtrate, and put the nucleic acid adsorption column back into the collection tube; (9) Add 400 μL of washing solution II to the nucleic acid adsorption column, centrifuge at room temperature, 12,000 rpm for 30 s, repeat this step once, discard the filtrate, and put the nucleic acid adsorption column back into the collection tube; (10) At room temperature, 12,000 rpm, centrifuge for 2 min to remove as much excess liquid as possible from the centrifuge column; (11) Transfer the nucleic acid adsorption column to a new 1.5 mL centrifuge tube, add 70 μL of RNase-Free Water to the middle of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge it at 12,000 rpm at room temperature for 2 min to obtain the RNA solution; (12) Remove the 1.5 mL collection tube with column from the centrifuge, check to make sure that the sample has been collected at the bottom of each tube, discard the column, and store the RNA sample at -80°C.
[0031] Comparative Example 1 The experimental process and experimental method are the same as those in Example 1, except that: The lysis buffer is: 100 mM Tris (pH 9.0), 150 mM NaCl, 50 mM EDTA, 1.5% sodium dodecyl sulfate (SDS), and 1.5% 2-mercaptoethanol (SDS and 2-mercaptoethanol were added before use).
[0032] Comparative Example 2 The experimental process and experimental method are the same as those in Example 1, except that: The lysis buffer was: 200 mM Tris, 250 mM Nacl, 50 mM EDTA and 4% PVP (added before use).
[0033] Example 2 The experimental process and experimental method are the same as those in Example 1, except that: RNA extraction from wheat roots: (2) Weigh 30 mg, 40 mg, and 50 mg of wheat roots respectively and put them into grinding tubes numbered 001, 002, and 003, and quickly put them into liquid nitrogen for temporary storage.
[0034] Comparative Example 3 The experimental process and experimental method are the same as those in Example 2, except that: The lysis buffer is: 100 mM Tris (pH 9.0), 150 mM NaCl, 50 mM EDTA, 1.5% sodium dodecyl sulfate (SDS), and 1.5% 2-mercaptoethanol; (SDS and 2-mercaptoethanol were added before use).
[0035] Comparative Example 4 The experimental process and experimental method are the same as those in Example 2, except that: The lysis buffer was: 200 mM Tris, 250 mM Nacl, 50 mM EDTA and 4% PVP (added before use).
[0036] Results Analysis (1) The experimental results of Example 1, Comparative Example 1 and Comparative Example 2 compare the quality of wheat seed RNA extracted under different lysis solutions, as shown in Tables 1 and Figure 1-3 shown.
[0037] Table 1 Quality of wheat seed RNA extracted from Example 1, Comparative Example 1 and Comparative Example 2
[0038] The results show that: in terms of RNA concentration, the lysate in Example 1 has a clear advantage; in terms of RQN, the 001 sample and the 002 sample in Example 1 and Comparative Example 1 are not much different, but the 003 sample is quite different, which shows that the lysate in Example 1 has good stability; in the peak graph, the RNA integrity after being treated by the lysate in Example 1 is significantly better. The quality of plant RNA extracted by the corresponding lysate solution scheme in this application is higher than that of the other two lysate solutions in all aspects.
[0039] (2) The experimental results of Example 2, Comparative Example 3 and Comparative Example 4 compare the quality of wheat root RNA extracted under different lysis solutions, as shown in Table 2 and Figure 4-6 shown.
[0040] Table 2 Quality of wheat root RNA extracted from Example 2, Comparative Example 3 and Comparative Example 4
[0041] The results showed that: in terms of RNA concentration, the lysis solution used in Example 2 had obvious advantages; in terms of RQN, the three samples of Example 2 were all above 7.0, which showed that the lysis solution in Example 2 had good stability; in the peak graph, the RNA integrity after being treated with the lysis solution in Example 2 was significantly better.
[0042] In summary, the quality of plant RNA extracted by the lysis solution used in this application is higher than that of the other two solutions in all aspects.
[0043] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.
Claims
1. A plant sample RNA extraction kit, characterized in that: It includes grinding materials, lysing solution, isopropanol, nucleic acid adsorption column, rinsing solution I, rinsing solution II and elution solution; The grinding material is a 3-6 mm steel ball; The lysis buffer includes pH 9.0, 80-100 mM Tris, 120-150 mM NaCl, 30-50 mM EDTA, 1.2-1.5% CTAB and 1.2-1.5% 2-mercaptoethanol; The rinse solution I comprises 500 g / L guanidine hydrochloride, 50% by volume ethanol, and 16% by volume nuclease-free water; The rinse solution II includes 50 mM Tris-HCl, 80% ethanol by volume, and 17.5% nuclease-free water by volume; The elution solution is nuclease-free water.
2. The kit according to claim 1, characterized in that: The abrasive materials include 4 mm steel balls and 6 mm steel balls.
3. The kit according to claim 1, characterized in that: The nucleic acid adsorption column is an RNA extraction column.
4. A method for stably extracting RNA from plant samples using the kit according to claims 1-3, characterized in that: The steps include: S1. Weigh the plant sample to be extracted, grind the material and place it in sample tube A, and pre-cool it in liquid nitrogen; S2. Add lysis buffer to pre-cooled sample tube A and shake to mix. Grind in a grinder at 4°C and 40 Hz for 20-30 seconds. After grinding thoroughly, centrifuge at 4°C and 10,000-12,000 rpm for 8-10 minutes, and transfer the supernatant to sample tube B. S3. Add isopropanol of the same volume as the supernatant solution to sample tube B and oscillate to mix; add the mixed solution in sample tube B to sample tube C assembled with a nucleic acid adsorption column, centrifuge at room temperature 10000-12000 rpm for 30 s, discard the filtrate, and reassemble the adsorption column back into sample tube C; S4, add rinse solution I to the sample tube C assembled with the nucleic acid adsorption column, centrifuge at room temperature, 10000-12000 rpm for 30 seconds, discard the filtrate, and reassemble the nucleic acid adsorption column back into the sample tube C; S5. Add 400 μL of washing solution II to the sample tube C assembled with the nucleic acid adsorption column, centrifuge at room temperature, 10000-12000 rpm for 30 s, and discard the filtrate; S6. Transfer the nucleic acid adsorption column to sample tube D, add 50-70 μL of nuclease-free water to the middle part of the adsorption membrane of the nucleic acid adsorption column, let it stand at room temperature for 2 min, centrifuge at 10000-12000 rpm at room temperature for 2 min, discard the nucleic acid adsorption column, and obtain the RNA solution.
5. The method according to claim 4, characterized in that: In the grinding step of step S2, after grinding once, the sample tube can be taken out and placed on dry ice to check the grinding condition of the sample. If the grinding is not sufficient, grinding materials with different particle sizes can be added for grinding again.
6. The method according to claim 4, characterized in that: After completing step S5, the nucleic acid adsorption column is reassembled into the sample tube C and centrifuged at room temperature, 10000-12000 rpm for 2 min to remove excess liquid in the nucleic acid adsorption column.
7. The method according to claim 6, characterized in that: In step S4, the rinsing liquid I is added for rinsing, which is repeated 1-2 times.
8. The method according to claim 7, characterized in that: In step S5, the rinsing liquid II is added for rinsing, which is repeated 1-2 times.
9. The method according to any one of claims 4 to 8, characterized in that: The sample tube A is a grinding sample tube, and the sample tubes B, C and D are EP tubes.
10. The method according to any one of claims 4 to 8, characterized in that: The plant samples to be extracted include roots, stems, leaves and seeds of plants.