A method for extracting total RNA from plateau plant Oxytropis glacialis
By combining liquid nitrogen grinding and two lysis solutions, the problems of low concentration and purity of RNA extraction from Oxytropis glacialis were solved, and high-quality RNA extraction was achieved, providing important sample support for the total RNA gene library and transcriptome sequencing of Oxytropis glacialis.
Patent Information
- Application Number
- CN202211126652.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-16
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2042-09-16
AI Technical Summary
Existing technologies make it difficult to extract high-concentration and high-purity RNA from Oxytropis glacialis, mainly because its leaf cell walls are relatively thick, resulting in poor RNA extraction results.
RNA was extracted using liquid nitrogen grinding combined with two lysis buffers. The first lysis buffer contained SDS, guanidine isothiocyanate, phenol, and sucrose, and the second lysis buffer contained EDTA, PVP, and NaCl. The combined use of the two lysis buffers inhibited endogenous RNase activity and chelated metal ions and polyphenol compounds, protecting RNA integrity.
High-concentration and high-purity RNA extraction was achieved, ensuring the stability and integrity of RNA and laying the foundation for subsequent molecular biology analysis.
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Figure CN115478070B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of molecular biology, and in particular to a method for extracting total RNA from a plateau plant, Oxytropis glacialis. Background Art
[0002] Oxytropis glacialis is a poisonous plant that contains a toxic alkaloid called swainsonine. Accidentally ingesting this plant can cause poisoning in animals, and there is currently no treatment available. Oxytropis glacialis has extremely short or stemless stems, making it highly resistant to the extreme conditions of the Qinghai-Tibet Plateau, such as hypoxia, high cold, and radiation. Compared to other plants, it can rapidly reproduce and spread in local environments, forming dominant communities there and posing a serious threat to ecological health and the development of animal husbandry. Toxicity analysis of Oxytropis glacialis is currently underway, with molecular biology as one of the research approaches. However, due to the thick cell walls of Oxytropis glacialis leaves, conventional plant RNA extraction techniques often yield low RNA concentrations and purities. Therefore, a new method for extracting high-quality RNA from Oxytropis glacialis is highly desirable. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for extracting total RNA from the plateau plant Oxytropis glacialis to solve the problems existing in the above-mentioned prior art. This method can extract RNA samples with higher concentration and purity, laying an important foundation for subsequent molecular biological analysis such as Oxytropis glacialis total RNA gene library and transcriptome sequencing.
[0004] To achieve the above object, the present invention provides the following solutions:
[0005] The present invention provides a method for extracting total RNA from a plateau plant, Oxytropis glacialis, comprising the following steps:
[0006] (1) Grinding the glacial oxtera material with liquid nitrogen, adding a first lysis solution to mix and lyse, and then centrifuging to obtain a supernatant; then adding a second lysis solution to the supernatant to lyse, and then centrifuging to obtain a supernatant;
[0007] (2) adding chloroform to the supernatant obtained in step (1) and mixing uniformly, then centrifuging to obtain the supernatant, adding isopropanol thereto, and centrifuging to obtain the precipitate;
[0008] (3) After rinsing with ethanol, the precipitate was dissolved in DEPC H2O and stored at low temperature;
[0009] The first lysis solution comprises 3-4% w / v SDS, 2-3% w / v guanidine thiocyanate, 40-50% v / v phenol, and 200-300 mmol / L sucrose;
[0010] The second lysis solution includes 20-30 mmol / L EDTA, 3-4% w / v PVP, and 200-250 mmol / L NaCl.
[0011] Furthermore, in step (1), the ratio of the first lysate and the second lysate to the glacial oxytropis material is 5 μl:1 mg.
[0012] Furthermore, in step (1), the pH of the first lysate and the second lysate are both adjusted to 6.0.
[0013] Furthermore, the adjustment is specifically carried out using 1M HCl.
[0014] Furthermore, the lysis condition in step (1) is standing on ice for 15 minutes, and the centrifugation is specifically 12000 rpm / min, 4°C, and centrifugation for 15 minutes.
[0015] Furthermore, in step (2), the volume ratio of the isopropanol to the supernatant is 1:1.
[0016] Furthermore, in step (3), before dissolving the precipitate in DEPC H2O, the ethanol in the precipitate needs to be sucked out and allowed to stand for 5 minutes.
[0017] Furthermore, the ethanol in step (3) is ethanol with a volume fraction of 75%.
[0018] The invention first grinds the glacial oxtera material with liquid nitrogen and fully homogenizes it, thus laying a foundation for subsequent full contact between the plant cells and the first lysis solution.
[0019] The components of the first lysis solution of the present invention function as follows: SDS is an anionic surfactant that denatures nuclear proteins and dissolves the phospholipid bilayer of the cell membrane, releasing RNA from the cell. Guanidine thiocyanate and phenol reagents strongly inhibit RNase, thereby ensuring the integrity of the extracted RNA. Sucrose is added to adjust the osmotic pressure of the solution to protect the RNA.
[0020] The functions of the components of the second lysis buffer: EDTA and PVP, as excellent chelating agents, can chelate metal ions and polyphenol compounds, effectively overcoming the effects of polyphenols and other substances on the RNA extraction process; and NaCl can ensure the stability of RNA.
[0021] The present invention uses two consecutive lysis steps. The first lysis solution is combined with the fully ground and homogenized raw materials to ensure RNA release while inhibiting endogenous RNase activity, making RNA less susceptible to degradation. An osmotic pressure regulator is also used to further protect RNA integrity. The second lysis solution uses an excellent chelating agent combined with polyphenols, metal ions and other substances to effectively avoid the impact of impurities on the RNA extraction process. By grinding the glacial oxtera material with liquid nitrogen and combining it with two consecutive lysis steps, the glacial oxtera leaf cells with thicker cell walls can be broken up, releasing a large amount of RNA. At the same time, the endogenous RNase activity is inhibited, and substances such as polyphenols are chelated to maximize the extraction of high-quality RNA with high concentration and purity.
[0022] The present invention discloses the following technical effects:
[0023] The present invention provides a method for extracting total RNA from the plateau plant Oxytropis glacialis based on the characteristic that the cell walls of the leaves are thick. The method grinds the plant material with liquid nitrogen to make it fully homogenized, laying the foundation for the subsequent plant cells to be fully contacted with the first lysate. The method adopts two consecutive lysis steps and is supplemented with the components of the lysate in a reasonable ratio. While ensuring the release of a large amount of RNA, the endogenous RNA enzyme activity is inhibited to avoid RNA degradation. The excellent chelating agent in the lysate can chelate metal ions and polyphenol compounds, effectively overcoming the effects of substances such as polyphenols on the RNA extraction process. At the same time, sucrose and NaCl osmotic pressure regulators are added to protect the integrity and stability of the RNA. The conventional RNA extraction method is used to extract the plateau plant Oxytropis glacialis RNA. The results show that the extracted RNA concentration is low, the RNA is degraded and the purity is not high, and the total RNA amount finally obtained is also relatively small, proving that the conventional RNA extraction method cannot obtain high-quality RNA from Oxytropis glacialis.
[0024] It can be seen that the present invention can maximize the extraction of RNA samples with higher concentration and purity by grinding plant materials with liquid nitrogen in combination with the first lysis solution and the second lysis solution in a reasonable ratio, laying an important foundation for subsequent molecular biology analysis such as the total RNA gene library and transcriptome sequencing of Oxytropis glacialis. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0026] Figure 1This is the gel electrophoresis diagram of the total RNA from the leaves of the plateau plant Oxytropis glaciense in Example 1, where M: RNA standard, 1: Yangbajing Town sample (latitude and longitude: 90.48799°E, 30.04268°N; 4287 meters above sea level), 2: Mount Everest Base Camp sample (latitude and longitude: 86.84309°E, 28.16793°N; 5003 meters above sea level), 3: Zabuye Salt Lake sample (latitude and longitude: 84.02377°E, 31.39431°N; 4453 meters above sea level), 4: Zhari Namucuo sample in Coqen County (latitude and longitude: 86.04237°E, 31.03991°N; 4710 meters above sea level). DETAILED DESCRIPTION
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0032] Unless otherwise specified, all reagents and instruments used in this example are commercially available conventional products. All reagents were prepared with DEPC water, sterilized at high temperature, and cooled for use.
[0033] Example 1
[0034] A method for extracting total RNA from the plateau plant Oxytropis glacialis, comprising the following steps:
[0035] (1) Preparation for extracting total RNA from Oxytropis glacialis leaves: Non-plastic utensils such as mortars and tweezers were wrapped in tin foil and sterilized by dry heat at 180°C for 6 h; plastic utensils were treated with 0.1% DEPC H2O, and the pipette tips were sterilized in an autoclave at a pressure of 103 kPa and 121°C for 30 min.
[0036] (2) First, the mortar and pestle were refrigerated with liquid nitrogen. Then, 100 mg of fresh Oxytropis glacialis material stored in RNALate was weighed (Oxytropis glacialis leaves from four different locations were selected as samples. Samples 1-4 in Table 1 were Yangbajing Town sample (latitude and longitude: 90.48799°E, 30.04268°N; altitude 4287 m), Mount Everest Base Camp sample (latitude and longitude: 86.84309°E, 28.16793°N; altitude 5003 m), Zabuye Salt Lake sample (latitude and longitude: 84.02377°E, 31.39431°N; altitude 4453 m) and Zha Ri Nam Co sample in Cuoqin County (latitude and longitude: 86.04237°E, 31.03991°N; altitude 4710 m)). After rinsing with H2O, place the sample in a mortar and add an appropriate amount of liquid nitrogen to thoroughly grind. Quickly transfer the ground material to a 2 ml RNase-free EP tube, add 500 μl of the first lysis buffer, mix thoroughly, and let stand on ice for 15 min. Centrifuge at 12,000 rpm / min, 4°C for 15 min, and collect the supernatant.
[0037] First lysis buffer: 3% w / v SDS, 2% w / v guanidine thiocyanate, 40% v / v phenol, 300 mmol / L sucrose; the pH of the lysis buffer was adjusted to 6.0 using 1 M HCl;
[0038] (3) Add 500 μl of the second lysis buffer, mix thoroughly, and let stand on ice for 15 minutes; centrifuge at 12000 rpm / min, 4°C for 15 minutes, and collect the supernatant;
[0039] Second lysis buffer: 20 mmol / L EDTA, 4% w / v PVP, 250 mmol / L NaCl; adjust the pH of the lysis buffer to 6.0 using 1 M HCl;
[0040] (4) Add 200 μl of chloroform, mix thoroughly, and let stand on ice for 15 minutes; centrifuge at 12,000 rpm / min, 4°C for 15 minutes, and collect the supernatant;
[0041] (5) Take 250 μl of the supernatant, add an equal volume of isopropanol (pre-cooled at -20°C), shake gently, and let it stand at -20°C for 15 minutes; centrifuge at 12000 rpm / min, 4°C for 10 minutes, and retain the precipitate;
[0042] (6) Add 1 ml of 75% ethanol to the sediment and rinse gently. Repeat twice, aspirate the alcohol, and place on ice for 5 minutes to evaporate the remaining alcohol.
[0043] (7) Add 30 μl of DEPC H2O to fully dissolve the RNA. Use Nanodrop 2000 (Thermo Scientific) to detect RNA concentration and purity. Then, use 1% agarose gel electrophoresis to detect RNA integrity (see Figure 1 ), the Oxytropis glacialis RNA samples that met the standards were labeled and quickly stored in a -80°C refrigerator for subsequent sequencing.
[0044] In order to extract higher quality total RNA from the plateau plant Oxytropis glacialis. The sample of Oxytropis glacialis material in step (2) of total RNA extraction from the plateau plant Oxytropis glacialis leaves is controlled to be around 100 mg, as too much material will cause endogenous RNase to degrade RNA; when taking the supernatant in step (3), the action should be gentle to prevent the Oxytropis glacialis RNA in the precipitate from being adsorbed, and all subsequent operations should be carried out on ice.
[0045] Example 2
[0046] The difference from Example 1 is that the first lysis solution contains: 4% w / v SDS, 3% w / v guanidine thiocyanate, 50% v / v phenol, and 200 mmol / L sucrose.
[0047] Example 3
[0048] The difference from Example 1 is that the second lysis solution contains: 30 mmol / L EDTA, 3% w / v PVP, and 200 mmol / L NaCl.
[0049] Comparative Example 1
[0050] The difference from Example 1 is that the conventional Trizol method is used to extract total RNA from Oxytropis glacialis.
[0051] Comparative Example 2
[0052] The difference from Example 1 is that the conventional CTAB method is used to extract total RNA from Oxytropis glacialis.
[0053] Comparative Example 3
[0054] The difference from Example 1 is that the conventional SDS method is used to extract total RNA from Oxytropis glacialis.
[0055] Effect Example 1
[0056] The RNA concentration and purity of each group were detected using Nanodrop 2000 (Thermo scientific) method. The results are shown in Table 1:
[0057] Table 1 Concentration and purity of total RNA extracted from Oxytropis glacialis in each group
[0058]
[0059] As shown in Table 1, the concentration of the total RNA of the plateau plant Oxytropis glacialis extracted by the method of the present invention exceeded 400 ng / μl, and the highest could reach 1064.5 ng / μl, which was relatively high. The ratio of A260 to A280 was between 1.8 and 2.2, and A260 / A230 was greater than 1.8, indicating that the RNA was not degraded and had a high purity. The total RNA finally obtained was greater than 7 ug, indicating that the quality of the obtained Oxytropis glacialis total RNA was high. Then, 1% agarose gel electrophoresis was used to detect whether the RNA was degraded. In the gel image of the extracted Oxytropis glacialis total RNA, the 18S band was bright and not degraded, indicating that the extracted Oxytropis glacialis total RNA was relatively complete (see Figure 1 ), it can be seen that the method of the present invention can be used to obtain total RNA from Oxytropis glacialis with higher concentration and purity.
[0060] In Comparative Examples 1-3, the total RNA concentration of the extracted plateau plant Oxytropis glacialis did not exceed 160 ng / μl, the concentration was relatively low, A260 / A280 <1.8 or >2.3, and A260 / A230 was less than 1.8, indicating that the RNA was degraded and the purity was not high. There may be contaminants in the sample, and the amount of total RNA obtained was also relatively small, proving that high-quality RNA from Oxytropis glacialis cannot be obtained using conventional RNA extraction methods.
[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for extracting total RNA from the plateau plant Oxytropis glacialis, characterized in that: The following steps are involved: (1) Grinding the glacial oxtera material with liquid nitrogen, adding a first lysis solution to mix and lyse, and then centrifuging to obtain a supernatant; then adding a second lysis solution to the supernatant to lyse, and then centrifuging to obtain a supernatant; (2) adding chloroform to the supernatant obtained in step (1) and mixing uniformly, then centrifuging to obtain the supernatant, adding isopropanol thereto, and centrifuging to obtain the precipitate; (3) After rinsing with ethanol, the precipitate was dissolved in DEPC H2O and stored at low temperature; The first lysis solution includes 3% w / v SDS, 2% w / v guanidine thiocyanate, 40% v / v phenol, and 300 mmol / L sucrose; The second lysis solution includes 20 mmol / L EDTA, 4% w / v PVP, and 250 mmol / L NaCl.
2. The method according to claim 1, characterized in that In step (1), the ratio of the first lysate and the second lysate to the glacial oxtera material is 5 μl:1 mg.
3. The method according to claim 1, characterized in that In step (1), the pH of the first lysate and the second lysate are both adjusted to 6.
0.
4. The method according to claim 3, characterized in that The adjustment is specifically performed using 1M HCl.
5. The method according to claim 1, wherein The lysis condition in step (1) is standing on ice for 15 minutes, and the centrifugation is specifically 12000 rpm, 4°C, and centrifugation for 15 minutes.
6. The method according to claim 1, characterized in that The volume ratio of the isopropanol to the supernatant in step (2) is 1:
1.
7. The method according to claim 1, characterized in that In step (3), before dissolving the precipitate in DEPC H2O, the ethanol in the precipitate needs to be aspirated and allowed to stand for 5 minutes.
8. The method according to claim 1, characterized in that The ethanol in step (3) is 75% by volume.
Citation Information
Patent Citations
Extracting method of leymus chinensis seeds RNA (Ribonucleic Acid) during early germination
CN108277218A