Method for extracting plant RNA (Ribonucleic Acid)

Abstract

The invention discloses a plant RNA extraction method, and belongs to the technical field of RNA extraction. The extraction method of the plant RNA comprises the following steps: mixing plant tissue powder, a CTAB lysate, a sulfydryl reducing agent and protease K, and then cracking at 60-65 DEG C to obtain a lysate; centrifuging the lysate, taking a supernatant, mixing the supernatant with a first solvent, oscillating, and washing the supernatant to obtain a precipitate; the precipitate and nuclease-free water are mixed and dissolved, and then DNase I and Buffer RDD are added for incubation; continuously adding sodium acetate, an RNA extraction phenol reagent and a second solvent, oscillating, carrying out ice bath, and centrifuging to obtain supernate; carrying out isopropanol precipitation on the supernate to obtain plant RNA (Ribonucleic Acid); the first solvent or the second solvent is a mixture of chloroform and isoamyl alcohol. According to the extraction method provided by the invention, the purpose of completely digesting the genome DNA is achieved on the premise of not influencing the quality of extracted RNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of RNA extraction, and particularly relates to a plant RNA extraction method. BACKGROUND

[0002] The currently popular method for plant RNA extraction is mainly CTAB method and various commercial polysaccharide polyphenol kits. However, various commercial kits are expensive and have poor extraction effect for some viscous samples. In comparison, CTAB method is widely used in plant RNA extraction and requires lower reagent price, and once configured, it can be used for several hundred times of extraction. Compared with commercial kits, CTAB method has lower cost and greater advantage.

[0003] However, the CTAB method requires higher freshness of the sample, and the conventional extraction process cannot completely digest the genomic DNA, which will affect the downstream qRT-PCR, RNA-seq and other experiments.

[0004] At present, the genomic DNA digestion process in the conventional CTAB method for extracting plant RNA mainly adds DNase I for digestion treatment together with the lysis solution at 65 DEG C lysis. However, the lysis time in the RNA extraction process is short, generally 5-10 min, and usually does not exceed 10 min. This will cause the genomic DNA released together to be not completely digested. Another digestion method is to centrifuge the lysis solution supernatant after 65 DEG C lysis, and then add DNase I (2.5 U / μL) in the supernatant and digest at 37 DEG C water bath for 25 min. This digestion method can achieve complete digestion of genomic DNA in terms of time, but since the lysis solution supernatant is generally about 800-900 μL, if it is completely digested, the required amount of DNase I (2.5 U / μL) is large, which greatly increases the extraction cost, is not conducive to large sample RNA extraction, and this digestion method cannot completely digest the genomic DNA due to the inability to quantitatively control the amount of DNase I used.

[0005] Therefore, the present application aims to improve the conventional CTAB method for extracting plant RNA and improve the conventional genomic DNA digestion procedure to completely digest the DNA contamination. SUMMARY

[0006] The present application aims to overcome the above technical deficiencies and provide a plant RNA extraction method to solve the technical problem that the genomic DNA cannot be completely digested in the prior art RNA extraction method.

[0007] To achieve the above technical purpose, the technical scheme of the present application provides a plant RNA extraction method, comprising the following steps: S1, mixing plant tissue powder, CTAB lysis solution, thiol reducing agent and proteinase K, and then lysing at 60-65°C to obtain a lysate; S2, centrifuging the lysate, taking supernatant, mixing the supernatant with a first solvent and oscillating, and then taking supernatant again, and washing the supernatant to obtain a precipitate; S3, dissolving the precipitate in nuclease-free water to obtain an RNA solution, and then adding DNase I and Buffer RDD and incubating; S4, continuing to add sodium acetate, RNA extraction phenol reagent and a second solvent and oscillating, and then ice-bathing, and then centrifuging to obtain supernatant; and precipitating the supernatant with isopropanol to obtain plant RNA; The first solvent or the second solvent is a mixture of chloroform and isopentanol.

[0008] In any embodiment, in step S1, the lysis time is 5-10 min.

[0009] In any embodiment, in step S1, the plant tissue includes one or more of leaves, roots, stems, flowers, fruits and seeds.

[0010] In any embodiment, in step S1, the plant is Impatiens walleriana or Scindapsus aureus.

[0011] In any embodiment, in the first solvent or the second solvent, the volume ratio of chloroform to isopentanol is (24-49):1.

[0012] In any embodiment, in step S3, the volume ratio of DNase I to the RNA solution is (2.5-3) μL:(100-120) μL.

[0013] In any embodiment, in step S3, the incubation is at 20-25°C for 20-25 min.

[0014] In any embodiment, in step S2, washing the supernatant to obtain a precipitate includes: adding pre-cooled isopropanol to the supernatant, mixing well, standing at -20 to -15°C, and then centrifuging and discarding the supernatant. Then, adding a pre-cooled 70-75% ethanol solution for washing, and discarding the ethanol to obtain the precipitate.

[0015] In any embodiment, in step S4, the oscillation time is 30-35 s.

[0016] In any embodiment, in step S4, the ice-bathing time is 5-10 min.

[0017] Compared with the prior art, the beneficial effects of the present application include: the extraction method proposed by the present application carries out nucleic acid precipitation twice, the first nucleic acid precipitation is to dissolve the mixture of DNA and RNA, reduce the digestion volume, and then use DNase I for digestion treatment, and the digestion can be carried out at room temperature 25℃, so as to reduce the damage of high temperature to nucleic acid. And this way can greatly reduce the use amount of DNase I (2.5 U / μL), and achieve the purpose of completely digesting genomic DNA without affecting the quality of extracted RNA. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the digestion result of genomic DNA extracted by the extraction method of embodiment 1 and comparative example 1 of the present application.

[0019] Figure 2 is the RNA electropherogram after the extraction method of embodiment 1 of the present application.

[0020] Figure 3 is the RNA electropherogram of different tissue parts after the extraction method of embodiment 1 of the present application. In order to verify the applicability of embodiment 1 to different tissue types of plants, the total RNA of the root, stem, leaf, flower and fruit tissue parts of Impatiens balsamina was also extracted, and it can be seen that the RNA bands of different tissue parts are very clear and almost no degradation. It shows that embodiment 1 is suitable for total RNA extraction of different tissue types of plants, and can make genomic DNA be completely digested. DETAILED DESCRIPTION

[0021] The "ranges" disclosed herein are defined by both a lower and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. Ranges defined by the limits of any of the foregoing can be either inclusive or exclusive of the end values, and can be arbitrarily combined, i.e., any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and 80-110 is listed for a particular parameter, it is understood that a range of 60-110 and 80-120 is also contemplated. Furthermore, if a minimum range value of 1 and 2 is listed, and if a maximum range value of 3, 4 and 5 is listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise stated, a numerical range "a-b" indicates a shorthand way of describing each and every intervening real number, i.e., a and b are both real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed herein, and "0-5" is merely a shorthand way of describing those numerical combinations. In addition, when it is stated that a parameter is an integer ≥ 2, it is equivalent to disclose that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0022] If not specifically stated, the "comprising" and "including" mentioned in the present application represent open-ended, and can also be closed-ended. For example, the "comprising" and "including" can represent that other components not listed can also be included, or only the listed components can be included.

[0023] If not specifically stated, in the present application, the term "or" is inclusive. For example, the phrase "A or B" represents "A, B, or both A and B". More specifically, any of the following conditions satisfy the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0024] The specific embodiment provides an extraction method of plant RNA, comprising the following steps: S1, mixing plant tissue powder, CTAB lysis solution, thiol reducing agent and proteinase K, and then lysing at 60-65°C for 5-10 min to obtain a lysate; the plant tissue includes one or more of leaves, roots, stems, flowers, fruits and seeds; in some embodiments, the plant is Impatiens balsamina or Scindapsus aureus; S2, centrifuging the lysate, taking the supernatant, mixing the supernatant with a first solvent and shaking, then taking the supernatant again, and washing the supernatant to obtain a precipitate; S3, dissolving the precipitate with water, then adding DNase I and Buffer RDD and incubating at 20-25°C for 20-25 min; S4, continuing to add sodium acetate, RNA extraction phenol reagent and a second solvent, shaking for 30-35 s, then ice-bathing for 5-10 min, and then centrifuging to obtain a supernatant; washing the supernatant to obtain plant RNA; The first solvent or the second solvent is a mixture of chloroform and isoamyl alcohol; the volume ratio of chloroform to isoamyl alcohol is (24-49):1. The main role of chloroform and isoamyl alcohol is to better remove protein contamination in the extraction of nucleic acids. Chloroform can denature proteins to separate them from nucleic acid solution. Isoamyl alcohol mainly acts as a defoaming agent to reduce the loss of nucleic acids.

[0025] In some embodiments, in step S3, the volume ratio of the DNase I to the RNA solution is (2.5-3) μL:(100-120) μL.

[0026] In some embodiments, in step S2, the washing of the supernatant to obtain the precipitate comprises: adding pre-cooled isopropanol into the supernatant, inverting and mixing, standing at -20 to -15°C, and then centrifuging and discarding the supernatant. Then, a pre-cooled ethanol solution with a mass concentration of 70-75% is added for washing, and the ethanol is discarded to obtain the precipitate.

[0027] The present application designs a twice-extraction and precipitation nucleic acid scheme, and after the first precipitation of nucleic acid, a genomic DNA digestion treatment is performed, which can use a normal temperature digestion condition to reduce the degradation of nucleic acid, and can also reduce the digestion volume, so as to reduce the use amount of DNase I (2.5 U / μL). The supernatant in the conventional CTAB extraction process is generally 700-900 μL, and the use amount of DNase I is generally 20 μL (the use amount of DNase I is 2.5 μL per 100 μL volume), and the improved digestion mode only needs 2.5 μL, directly reducing the use amount of DNase I by 87.5%.

[0028] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0029] In the present application, "some embodiments", "the present embodiment" and examples and the like are described, which describe a subset of all possible embodiments, but it can be understood that "some embodiments" can be the same subset or different subset of all possible embodiments, and can be combined with each other without conflict.

[0030] If the application file contains similar descriptions of "first / second", the following description is added, in the following description, the terms "first\second\third" are only used to distinguish similar objects, and do not represent a specific order of the objects, and it can be understood that "first\second\third" can be interchanged in a specific order or sequence as allowed, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0031] In the present embodiment, the term "and / or" is only used to describe the association relationship of the associated objects, which means that there can be three relationships, for example, object A and / or object B, which can mean that object A exists alone, object A and object B exist together, and object B exists alone.

[0032] The following describes the examples of the present application. The examples described below are exemplary and are for the purpose of explanation of the present application and cannot be understood as a limitation of the present application. In the examples, the specific techniques or conditions not noted are performed according to the techniques or conditions described in the literature in the field or according to the product manual. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained commercially.

[0033] Example 1 This example proposes a method for extracting plant RNA, comprising the following steps: 1.1 Grinding and lysing of tissue samples Pre-treatment of mortar: Wrap the mortar, pestle and spoon with tin foil, pour in an appropriate amount of anhydrous ethanol, and ignite. Note that the burning treatment should be away from the smoke alarm, and the whole process needs to be in the presence of the person.

[0034] a) Take 0.1-0.2 g of plant tissue (leaf, root, stem, flower, fruit or seed sample) and put it into a mortar that has been added with liquid nitrogen, grind quickly until the sample is broken into powder; The weight of the sample tube or tin foil should be weighed before and after sampling to calculate the sampling amount. Reference: 0.05-0.1 g of tissue can extract 5-10 μg of RNA, and the sampling amount should be increased to 0.2 g for stems and seeds due to the lower extraction rate; b) Store the remaining sample in a -80℃ refrigerator; c) Transfer the powder ground in the mortar to a 2 mL centrifuge tube, add 1 mL of 2% CTAB lysis solution, 4 μL of thiol reducing agent, and 10 μL of proteinase K (20 mg / mL) to the 2 mL centrifuge tube, mix well, and then place it in a constant temperature water bath at 65℃ for 5-10 min (until no obvious tissue is visible); d) Centrifuge the lysed sample at 12000 rpm for 2 min; e) Take 700 μL of supernatant to a new 2 mL centrifuge tube; 1.2 24:1 extraction and first isopropanol precipitation (Note: performed in a fume hood) a) Add 1 volume of the first solvent chloroform:isopentanol (24:1) to the supernatant obtained in the above step, shake for 30 s, and then centrifuge at 12000 rpm for 5 min; b) Transfer the supernatant to a new 2 mL centrifuge tube, making sure not to suck up the middle protein layer; c) Add an equal volume of pre-cooled isopropanol to the supernatant of the previous step, mix well by inverting, and stand at -20℃ for 10 min; d) Centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant; e) Add 1 mL of 75% ethanol (RNA) pre-cooled at -20°C, and discard the ethanol.

[0035] 1.3 DNase I digestion of DNA a) Add 87.5 μL of RNA-free water to dissolve the RNA; b) Add 2.5 μL of DNase I and 10 μL of Buffer RDD to obtain 100 μL of RNA solution, mix gently, and incubate at room temperature (25°C) for 20 min; 1.4 Secondary extraction and purification (Note: perform in a fume hood) a) Add 0.1 times the volume of 3 mol / L sodium acetate (pH 5.2, to keep the solution acidic) to the supernatant from the previous step; b) Add 1 times the volume of RNA extraction phenol reagent (use after standing, take the lower organic phase. The upper layer is a citric acid solution, which prevents oxidation of the lower organic phase) to the supernatant from the previous step; c) Add 200 μL of the second solvent chloroform:isopropyl alcohol (49:1), shake for 30 s, and then ice bath for 5 min; d) Centrifuge at 12000 rpm for 5 min; e) Transfer the supernatant to a new 1.5 mL centrifuge tube, being careful not to suck up the middle protein layer; 1.5 RNA precipitation and washing a) Add an equal volume of pre-cooled isopropyl alcohol to the supernatant from the previous step, mix well by inverting, and stand at -20°C for 10 min; b) Centrifuge at 12000 rpm for 10 min, and discard the supernatant; c) Add 1 mL of 75% ethanol (RNA) pre-cooled at -20°C, and discard the ethanol; repeat the washing once, and dry the tube opening with a filter paper; d) Lightly centrifuge the tube on a mini centrifuge, use a pipette to suck up the residual liquid in the tube, and dry at room temperature for 2-5 min, until the ethanol is completely dry; e) Add 20-30 μL of RNA-free water to dissolve the RNA. The RNA yields of different tissues obtained by this method are shown in Table 1.

[0036] Table 1 RNA yields of different tissue parts extracted in Example 1 As can be seen from Table 1, the RNA quality of different tissues of Impatiens balsamina extracted is good overall, the RNA concentration and integrity of fruits and flowers are optimal, followed by leaves, and roots and stems are moderate. As can be seen from the 2100 RIN value, the RNA integrity is very good, and the RIN value can be as high as 9.5.

[0037] Comparative Example 1 This comparative example extracts plant RNA by the existing CTAB method, specifically, 1.1 Grinding and lysis of tissue samples a) Take an appropriate amount of green leaves and put them into a mortar that has been added with liquid nitrogen, grind quickly until the sample is broken into powder; b) Transfer the powder ground in the mortar to a 2 mL centrifuge tube, add 1 mL of 2% CTAB lysis solution, 4 μL of thiol reducing agent, and 10 μL of proteinase K (20 mg / mL) to the 2 mL centrifuge tube, mix well, and then place in a constant temperature water bath at 65°C for lysis for 5-10 min (until no obvious tissue is observed); c) Centrifuge the lysed sample at 12000 rpm for 2 min, and take 700 μL of supernatant to a new 2 mL centrifuge tube; d) Add 100 μL of DNase I working solution (20 μL of DNase I and 80 μL of Buffer RDD) to the supernatant of the previous step, mix well, and then incubate in a water bath at 37°C for 15 min.

[0038] 1.2 Extraction and purification (Note: performed in a fume hood) a) Add 0.1 times the volume of 3 mol / L sodium acetate (pH 5.2) and 1 times the volume of chloroform:isopropyl alcohol (24:1) to the supernatant of the previous step, mix well, and then centrifuge at 12000 rpm for 5 min; b) Transfer the supernatant to a new 2 mL centrifuge tube, making sure not to suck up the middle protein layer; c) Add an equal volume of chloroform:isopropyl alcohol (24:1) to the supernatant of the previous step, mix well, and then centrifuge at 12000 rpm for 5 min; d) Transfer the supernatant to a new 1.5 mL centrifuge tube; 1.3 RNA precipitation and washing a) Add an equal volume of pre-cooled isopropyl alcohol to the supernatant of the previous step, mix well by inverting, and then stand at -20°C for 10 min; b) Centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant; c) Add 1 mL of -20°C pre-cooled 75% ethanol (RNA) for washing, and discard the ethanol. Repeat the washing once, and then use a water absorption paper to absorb the residual liquid in the tube; d) Lightly centrifuge on a mini centrifuge, use a pipette to suck the residual liquid in the tube, and then place on a Thermomixer for drying at 37°C for 2 min; e) Dissolve the RNA in 10-30 µL of RNA free-Water, and store at -80°C for long-term preservation.

[0039] Combine Figure 1 ,from Figure 1 It can be seen that in Comparative Example 1, when total RNA was extracted from Scindapsus leaves using the conventional CTAB method, genomic DNA was precipitated together with the RNA. The normal digestion process can remove some DNA contamination, but the DNA cannot be completely digested. However, in Example 1, when the modified CTAB method was used for extraction, it can be seen that the genomic DNA was completely digested after the addition of DNase I.

[0040] Figure 2 This is a validation of the large-scale extraction using the modified CTAB method in Example 1, using leaves of Scindapsus radiata as the extract. As can be seen from the figure, the electrophoretic bands for all 23 RNA species are clear and well-intact. Only 15 channels have a small amount of residual DNA, likely due to the large sample size. Overall, however, genomic DNA contamination was completely removed.

[0041] Combine Figure 3 To verify the applicability of Example 1 to different plant tissue types, total RNA was extracted from the roots, stems, leaves, flowers, and fruits of Impatiens balsamina. Clear RNA bands were observed across these tissues, with minimal degradation. This demonstrates that Example 1 is suitable for total RNA extraction from various plant tissue types and results in complete digestion of genomic DNA. M in the figure represents the Trans2K Plus II DNA Marker, and the size distribution of electrophoretic bands can be determined by comparing M.

[0042] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for extracting plant RNA, characterized in that: The following steps are involved: S1. Mix plant tissue powder, CTAB lysis buffer, sulfhydryl reducing agent, and proteinase K, and then lyse at 60-65°C to obtain a lysate; S2. Centrifuging the lysate, taking the supernatant, mixing the supernatant with the first solvent and shaking, then taking the supernatant again, and washing the supernatant to obtain a precipitate; S3, mixing the precipitate with nuclease-free water to dissolve it to obtain an RNA solution, and then adding DNaseI and Buffer RDD for incubation; S4, continue to add sodium acetate, RNA extraction phenol reagent and the second solvent, shake, ice bath, and then centrifuge to obtain a supernatant; precipitate the supernatant with isopropanol to obtain plant RNA; The first solvent or the second solvent is a mixture of chloroform and isoamyl alcohol.

2. The method for extracting plant RNA according to claim 1, wherein In step S1, the lysis time is 5-10 minutes.

3. The method for extracting plant RNA according to claim 1, wherein In step S1, the plant tissue includes one or more of leaves, roots, stems, flowers, fruits and seeds.

4. The method for extracting plant RNA according to claim 3, wherein In step S1, the plant is Impatiens balsamina or Scindapsus scindapsus.

5. The method for extracting plant RNA according to claim 1, wherein In the first solvent or the second solvent, the volume ratio of chloroform to isoamyl alcohol is (24-49):

1.

6. The method for extracting plant RNA according to claim 1, wherein In step S3, the volume ratio of the DNaseI to the RNA solution is (2.5-3) μL:(100-120) μL.

7. The method for extracting plant RNA according to claim 1, wherein In step S3, the mixture is incubated at 20-25°C for 20-25 minutes.

8. The method for extracting plant RNA according to claim 1, wherein In step S2, washing the supernatant to obtain a precipitate includes: adding pre-cooled isopropanol to the supernatant, mixing by inversion, standing at -20 to -15°C, centrifuging and discarding the supernatant, and then adding pre-cooled ethanol solution to wash the precipitate.

9. The method for extracting plant RNA according to claim 1, wherein In step S4, the oscillation time is 30-35 seconds.

10. The method for extracting plant RNA according to claim 1, wherein In step S4, the ice bath time is 5-10 minutes.

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