CN1727357A - Method for Extracting RNA from Cotton Tissue - Google Patents

Abstract

A process for extracting RNA from cotton tissue includes such steps as extracting in guanidine thiocyanate-chloroform solution, dissolving the coarse extract in the cracking liquid containing the polyvinyl pyrrolidone 4000 and beta-mercaptoethanol, and purifying by water-saturated phenol.

Description

Method for Extracting RNA from Cotton Tissue

technical field

The invention relates to the technical field of cotton molecular biology. Specifically, it relates to a method for extracting and purifying RNA used in molecular biology from cotton tissue.

Background technique

RNA extraction is the basis for molecular biology research. The acquisition of high-quality RNA is of great significance for functional genomics and genetics research. There are many extraction methods for plant RNA, the existing methods mainly include guanidinium thiocyanate-phenol-chloroform extraction and purification method, phenol-SDS method and CTAB method, etc., wherein guanidine thiocyanate-phenol-chloroform The extraction and purification method is widely used in the extraction of RNA from various animals and plants, and is an effective and economical method for extracting RNA. Cotton is a kind of shrub woody plant. Its tissues and organs are rich in polyphenols, polysaccharides and secondary metabolites. It is difficult to apply extraction methods such as extracting DNA, RNA and protein according to traditional methods on cotton.

In recent years, the separation and purification of cotton biological macromolecules (such as DNA, RNA and protein, etc.) has attracted the attention of scientists and technicians in the field of molecular biology technology. The improvement of DNA extraction methods has promoted the development of cotton molecular biology at the DNA level. In recent years, there have been many reports on the methods of extracting RNA from cotton tissues and organs at home and abroad, such as using the improved thermal boron method (YingRu Wu et al., Plant Molecular Biology Reporter 20: 213-218; Li Jigang et al., Cotton Journal , 2004, 16(1): 3～7); CTAB method (Jiang Jianxiong et al., Acta Cotton Sinica, 2003, 15(3): 166～167; 150), phenol SDS method (Xia Lanqin et al., Acta Cotton Sinica, 2000, 12(4): 205-207), etc. The improved thermal boron method combines the crude extraction of the thermal boron method and the purification of the RNA purification kit to ensure the quality of the RNA molecular manipulation in the future. However, the extraction cost of this method is relatively high. Jiang Jianxiong et al., Li Jigang et al., Xia Lanqin et al. used the improved CTAB method, hot boron method and phenol-SDS method to extract the total RNA of cotton tissue respectively. Improve the quality of RNA samples, so the whole extraction process (time) is longer. Because the extraction time is too long, the possibility of RNA degradation will be greatly increased.

The RNA extraction method of guanidinium thiocyanate-phenol-chloroform is widely used in many plants, but many scholars such as Jiang Jianxiong, Xu Yanong, Xia Lanqin found that the traditional guanidine thiocyanate-phenol-chloroform extraction The method cannot or is not suitable for RNA extraction in cotton (Jiang Jianxiong et al., Acta Cotton Sinica, 2003, 15(3): 166-167; Xia Lanqin et al., Acta Cotton Sinica, 2000, 12(4): 205-207). According to the physiological and biochemical characteristics of secondary substances (such as polyphenols) in cotton, Wang Wenfeng et al. proposed to use high pH value instead of traditional low pH value to improve the guanidine thiocyanate-phenol-chloroform extraction method to adapt to cotton tissue. The application of high pH value has a certain effect on the oxidation of cotton polyphenols, but DNA and RNA exist in the buffer solution with high pH value during the extraction process, and DNA needs to be further removed.

As we all know, the influence of polyphenols, polysaccharides and secondary metabolites in cotton, compared with other plants such as rice, rape and other crops, will encounter more difficulties in the process of RNA extraction from polyphenol-rich cotton. This is mainly manifested in the oxidation of polyphenols, the co-precipitation of polysaccharides and nucleic acids, the removal of secondary metabolites and proteins, etc. Polyphenols are easily oxidized after cell rupture and form derivatives such as quinones, which can irreversibly bind to nucleic acids and co-precipitate with nucleic acids or be extracted by organic solvents such as chloroform and phenol. This will greatly affect the quality and quantity of RNA and form an obstacle to future related molecular biology operations. Due to unknown factors, phenol cannot be used to remove polysaccharides and proteins at the beginning of DNA and RNA extraction. Therefore, the traditional guanidinium thiocyanate-phenol-chloroform extraction and purification method cannot be effectively applied to cotton. However, the ribonucleic acid obtained by chloroform extraction alone contains more polysaccharides and proteins, which affects the quality of ribonucleic acid. At the same time, due to the lack of purification by phenol, the nucleic acid obtained by utilization will also contain secondary metabolites, which may be the reason for the low efficiency of subsequent molecular operations such as reverse transcription of RNA. Due to the limitations of these objective factors, obtaining high-quality nucleic acid has become the primary problem to be solved in cotton molecular biology research.

The guanidine thiocyanate generally adopted in the existing technology of extracting RNA from cotton tissue--phenol extraction--chloroform extraction order is not suitable for the extraction of cotton tissue RNA, adopts prior art, in phenol extraction RNA is easily lost during the process, which cannot satisfy the molecular biology research of cotton.

Contents of the invention

The purpose of the present invention is to overcome the defect of prior art, establish a kind of new method of extracting and improving quality RNA from cotton tissue, this method adopts guanidinium thiocyanate-chloroform to extract, and phenol purifies after sample precipitation and dissolving , to obtain high-quality RNA samples from cotton tissues. The method of the invention is fast and economical, and is suitable for cotton molecular biology operations. By using the method, the tissue sample can be stored in the lysate for a long time at the same time, and the RNA sample can be better guaranteed not to be degraded.

The inventor has developed a method for extracting RNA from cotton tissue that satisfies the purpose of the present invention through a large number of research comparisons and experiments. It includes the following steps:

(1) Homogenization: take 1 weight part of cotton tissue, wash it with clear water or directly grind it into powder in liquid nitrogen or a glass homogenizer, and the tissue material is mixed with the pre-mixture in a volume/weight ratio of 1:5-15. Mix cold RNA extraction buffer (pH 6.0-6.5) and add 0-0.2 parts of β-mercaptoethanol and 0.8-1.2 volumes of sodium acetate (pH 5.0-5.2) to mix by inverting and place on ice , to obtain the sample mixture;

(2) Add an equal volume of chloroform to the sample mixture obtained in step (1), turn it upside down to make it evenly mixed into one phase, place it on ice for 10-15 minutes, then centrifuge at 4°C for 15 minutes, and recover the supernatant;

(3) Take the supernatant of step (2), add an equal volume of frozen (-20°C) isopropanol, mix upside down, place at -20°C for 0.5-1 hour to precipitate RNA, and centrifuge at 4°C for 15 Minutes to get sediment;

(4) Dissolve the precipitate obtained in step (3) thoroughly with 1-5 parts by volume of RNA extraction buffer (pH6.0-6.5), then add 1-5 parts by volume of acid phenol solution (pH 4.7-5.2) , mix upside down, ice bath for 10-15 minutes, centrifuge at 4°C for 15 minutes, and recover the supernatant;

(5) Add 1-5 parts by volume of chloroform solution to the supernatant in step (4), mix well and ice-bath for 10-15 minutes, centrifuge at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.0-5.2) and 2.5-3 times the volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 0.5-1 hours to precipitate the RNA, and centrifuge at 4°C for 15 minutes to obtain the precipitate;

(7) Wash the precipitation of step (6) 2-3 times with 75% ethanol, then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

Wherein: the RNA extraction buffer is to use 472.64 grams of guanidine thiocyanate, 7.352 grams of sodium citrate, 5-10 grams of sodium lauryl sarcosine, 0.5-20 grams of polyvinylpyrrolidone 4000 ( PVP4000) is obtained by adding water to 950 ml, adjusting the pH to 6.0-6.5, adding water to dilute to one liter and extracting;

Described sodium acetate solution is to dissolve with the sodium acetate of 408 grams in water, adjust pH 5.0-5.2 with glacial acetic acid and add water to settle to one liter, obtain after autoclaving;

The centrifugation is 9000×g-12000×g;

Described chloroform solution is that the chloroform of 24 volume parts and the isoamyl alcohol of 1 volume part mix;

The acid phenol solution (pH4.7-5.2) is formed by mixing 1 volume part of water-saturated phenol (pH4.7-5.2) and 1 volume part of the above-mentioned chloroform solution;

The 75% ethanol is mixed with 3 parts of absolute ethanol and 1 part of water;

The water is pure water treated with 0.1% DEPC and sterilized by high pressure.

Preferably, the used cotton tissue material is fresh and healthy, and the root tissue should be washed with clear water first.

The beneficial effects of the present invention are:

The present invention has done a lot of improvement work to traditional guanidine thiocyanate-phenol-chloroform RNA extraction method:

Polyvinylpyrrolidone 4000 is added to the RNA extraction buffer, and its dosage can be adjusted appropriately according to the content of polyphenols in the material, and β-mercaptoethanol is used to effectively prevent the oxidation of polyphenols.

After the tissue sample is mixed with the RNA extraction buffer, it can be stored in an ultra-low temperature refrigerator for a long time and ensure the integrity of the RNA, which solves the limitation that RNA can only use fresh tissue.

After using chloroform solution to extract the sample mixture for the first time, use isopropanol to precipitate the crude RNA extract, which effectively reduces the volume of the solution in subsequent operations, and reduces The content of secondary metabolites in the solution.

Different from the traditional guanidine thiocyanate-phenol-chloroform, the innovation of the present invention is that it is first extracted with chloroform, then precipitated with isopropanol to obtain the crude extract RNA, and then purified by extraction with acid phenol and chloroform, and finally purified with Ethanol precipitation also effectively reduces the co-precipitation of polysaccharides and improves the quality of RNA.

The present invention is applicable to different tissues of cotton. The present invention has been used to successfully extract high-quality RNA from cotton leaves (such as cotyledons, true leaves), roots, callus, flowers (petals or flower buds), hypocotyls, fibers and other tissues.

The invention is economical and efficient. Most of the medicines used in the present invention are common biochemical reagents, which are cheap and do not utilize kits or expensive medicines. In terms of time, there is no overnight precipitation or long-term treatment, and generally all experiments can be completed within one working day.

Description of drawings

The electrophoresis (1X TAE, 1.5%) of the total RNA extracted from the root after Fig. 1 sea island cotton seedlings inoculated with Verticillium dahliae on non-denatured agarose gel

Figure 2 Electrophoresis of total RNA from different cotton tissues on denatured agarose gel (1x MOPS, 1.5%). The sources of tissues in the figure are: 1-6 are roots, 7-8 are hypocotyls, and 9-12 are true leaves.

Figure 3 Electrophoresis of total cDNA in cotton roots. 1 is the total cDNA, M is the molecular weight standard (#SM0321, MBI).

Fig. 4 Electrophoresis of insert fragments of partial clones of root full-length cDNA library. 1 to 32 in the figure are different cloned cDNA inserts, and M is the molecular weight standard (#SM0321, MBI).

Fig. 5 RT-PCR and genomic PCR detection of cotton disease resistance related gene (AY560551). Samples 1-10 are cDNA templates, and sample 11 is genomic DNA template; M is molecular weight standard (#SM0321, MBI).

Fig. 6 The results of northern blotting of the cotton nematode-induced expression gene MIC-3 induced by Verticillium dahliae. 1 is the cotton control not inoculated with the disease source; 2-9 are the expression of cotton at 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after the cotton was inoculated with Verticillium wilt.

Detailed ways

Embodiment 1: utilize cotton root to extract RNA sample

(1) Take 2 grams of fresh cotton root, wash it with water and grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 20 ml of pre-cooled RNA extraction buffer (pH6.0 ) (containing 0.5% PVP4000) and 200 microliters of β-mercaptoethanol, upside down to mix evenly, add 2 ml of sodium acetate, mix evenly;

(2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, place it in a -20°C refrigerator for 30 minutes, and centrifuge at 10,000×g for 15 minutes at 4°C;

(4) Discard the supernatant, dissolve the precipitate completely with 3ml RNA extraction buffer, then add 3ml of acid phenol solution (pH 4.7-5.2), mix upside down, ice bath for 15 minutes, 4°C, 12000× Centrifuge at g for 15 minutes, and recover the supernatant;

(5) Add 3 ml of chloroform solution to the supernatant in step (4), mix well and ice bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.0) and 2.5 times volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 0.5 hours To precipitate RNA, centrifuge at 10,000×g for 15 minutes at 4°C to obtain a precipitate;

(7) washing with 75% ethanol to wash the precipitation of step (6) twice, and then place it at room temperature for 5-10 minutes to dry it, so that the pure RNA of the present invention can be obtained.

Welcome to use this embodiment, the total amount of cotton RNA sample obtained is 820 micrograms, and the yield is 410 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.85, meeting the needs of molecular biology experiments.

Embodiment 2: utilize cotton true leaf to extract RNA sample

(1) Take 1 gram of fresh cotton true leaves, grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 15 ml of pre-cooled RNA extraction buffer (pH6.5) (containing 2 %PVP4000) and 200 microliters of β-mercaptoethanol, upside down to mix evenly, add 1 ml of sodium acetate, mix evenly;

(2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant from step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 1 hour, 4°C, 12000×g Centrifuge for 15 minutes to recover the precipitate;

(4) Discard the supernatant, dissolve the precipitate completely with 5ml RNA extraction buffer, then add 5ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 15 minutes, 4°C, 12000× Centrifuge at g for 15 minutes, and recover the supernatant;

(5) Add 5 ml of chloroform solution to the supernatant in step (4), mix well and ice bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH5.2) and 3 times the volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 0.5 hours to precipitate the RNA, and centrifuge at 12000×g for 15 minutes at 4°C to obtain the precipitate;

(7) Wash the precipitation of step (6) with 75% ethanol for 3 times, and then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

The total amount of cotton RNA sample obtained by applying this embodiment is 730 micrograms, and the yield is 730 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.74, which meets the needs of molecular biology experiments.

Embodiment 3: extract RNA sample from cotton callus

(1) Get 0.3 grams of fresh cotton callus and put it into a glass homogenizer, and add 3 milliliters of pre-cooled RNA extraction buffer (pH6.3) (containing 0.5% PVP4000), grind it, and add 0.24 milliliters of Sodium acetate, mix well, pour into the centrifuge tube;

(2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 10 minutes, centrifuge at 12000×g for 10 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant from step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 0.5 hours, 4°C, 10000×g Centrifuge for 10 minutes to recover the precipitate;

(4) Discard the supernatant, dissolve the precipitate thoroughly with 0.5ml RNA extraction buffer, then add 0.5ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 10 minutes, 4°C, 12000 Centrifuge at × g for 15 minutes, and recover the supernatant;

(5) Add 0.5 ml of chloroform solution to the supernatant in step (4), mix well and ice-bath for 10 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.0) and 2.5 volumes of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 1 hour To precipitate RNA, centrifuge at 10,000×g for 15 minutes at 4°C to obtain a precipitate;

(7) washing with 75% ethanol to wash the precipitation of step (6) twice, and then place it at room temperature for 5-10 minutes to dry it, so that the pure RNA of the present invention can be obtained.

The total amount of cotton RNA sample obtained by applying this embodiment is 338 micrograms, and the yield is 1014 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.92, meeting the needs of molecular biology experiments.

Embodiment 4: extract RNA sample from cotton flower

(1) Take 0.8 g of fresh cotton petals, grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 12 ml of pre-cooled RNA extraction buffer (pH6.4) (containing 2% PVP4000), add 160 microliters of β-mercaptoethanol, turn it upside down to mix evenly, add 0.9 ml of sodium acetate, and mix evenly;

(2) Add an equal volume of chloroform solution, mix it upside down to make it a homogeneous phase, place it on ice for 15 minutes, centrifuge at 12000×g for 10 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant from step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 0.5 hours, 4°C, 10000×g Centrifuge for 15 minutes to recover the precipitate;

(4) Discard the supernatant, dissolve the precipitate completely with 6ml RNA extraction buffer, then add 6ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 15 minutes, 4°C, 12000× Centrifuge at g for 15 minutes, and recover the supernatant;

(5) Add 6 ml of chloroform solution to the supernatant in step (4), mix well and ice bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.2) and 3 times volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 1 hour To precipitate RNA, centrifuge at 10,000×g for 15 minutes at 4°C to obtain a precipitate;

(7) Wash the precipitation of step (6) with 75% ethanol for 3 times, and then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

The total amount of cotton RNA sample obtained by applying this embodiment is 435 micrograms, and the yield is 544 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, A _260/280 = 1.83, meeting the needs of molecular biology experiments.

Embodiment 5: extract RNA sample from the hypocotyl of cotton

(1) Take 0.2 g of fresh cotton hypocotyl, put it into a glass homogenizer, and add 3 ml of pre-cooled RNA extraction buffer (pH6.5) (containing 1% PVP4000) and 40 microliters of β-mercapto Ethanol, grind it, add 0.2 ml of sodium acetate, mix well, and pour it into a centrifuge tube;

(2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant obtained in step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 0.5 hours, 4°C, 12000× Centrifuge at g for 15 minutes to recover the precipitate;

(4) Discard the remaining supernatant in step (3), dissolve the precipitate thoroughly with 0.6ml RNA extraction buffer, then add 0.6ml of acid phenol solution (pH 4.7-5.2), mix up and down, and ice bath for 15 Minutes, centrifuge at 12,000×g for 15 minutes at 4°C, and recover the supernatant;

(5) Add 0.6 ml of chloroform solution to the supernatant obtained in step (4), mix well and ice-bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(7) Add 1/10 volume of sodium acetate (pH 5.0) and 3 times volume of absolute ethanol to the supernatant obtained in step (5), mix well, and place at -20°C for 0.5 hours to precipitate the RNA, and centrifuge at 10,000×g for 15 minutes at 4°C to obtain the precipitate;

7 Wash the precipitation of step (6) with 75% ethanol for 3 times, and then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

Applying this embodiment, the total amount of cotton RNA sample obtained is 95 micrograms, and the yield is 475 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.78, which meets the needs of molecular biology experiments.

Embodiment 6: extract RNA sample from fresh cotton fiber

(1) Strip 0.2 g of cotton fiber 10 days after pollination from fresh cotton bolls, put it into a glass homogenizer, and add 3 ml of pre-cooled RNA extraction buffer (pH6.4) (containing 1% PVP4000) and 30 microliters of β-mercaptoethanol, grind it, add 0.2 milliliters of sodium acetate, mix well, and pour it into a centrifuge tube;

(2) Add an equal volume of chloroform solution, mix it upside down to make it a uniform phase, place it on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and collect the supernatant;

(3) Take the supernatant of step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 0.5 hours, 4°C, 12000×g Centrifuge for 15 minutes to recover the precipitate;

(4) Discard the supernatant, dissolve the precipitate thoroughly with 0.6ml RNA extraction buffer, then add 0.6ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 15 minutes, 4°C, 12000 Centrifuge at × g for 15 minutes, and recover the supernatant;

(5) Add 0.6 ml of chloroform solution to the supernatant in step (4), mix well and ice-bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.1) and 2.5 times volume of absolute ethanol to the supernatant of step (5) and mix well, and place it at -20°C for 0.5 hours to Precipitate RNA, centrifuge at 10,000×g at 4°C for 15 minutes to obtain the precipitate;

(7) Wash the precipitation of step (6) with 75% ethanol for 3 times, and then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

Applying this embodiment, the total amount of RNA obtained was 96 micrograms, and the yield was 480 micrograms/gram

Quality inspection: RNA remained intact, A _260/280 = 1.88, meeting the needs of molecular biology experiments.

Embodiment 7: extract RNA sample from cotton seedling

(1) Take 3 grams of fresh cotton seedling whole plant, grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 45 ml of pre-cooled RNA extraction buffer (pH6.2) (containing 2% PVP4000) and 600 microliters of β-mercaptoethanol, upside down to mix evenly, add 4.5 ml of sodium acetate, mix evenly;

(2) Divide the mixture in step (1) into two equal parts, put each part into a new tube, add an equal volume of chloroform solution to each tube, mix it upside down to make it a uniform phase, and then Place on ice for 15 minutes, centrifuge at 12,000×g for 15 minutes at 4°C, and recover the supernatant;

(3) Take the supernatant from the two tubes of step (2) into two new tubes, add an equal volume of frozen (-20°C) isopropanol to precipitate the RNA, and place it in a -20°C refrigerator for 1 hour at 4°C. , centrifuge at 12000×g for 15 minutes, and recover the precipitate;

(4) Discard the supernatant, dissolve each tube of precipitation thoroughly with 7.5ml RNA extraction buffer, combine the two tubes of solution into one tube, add 15ml of acid phenol solution (pH 4.7-5.2), mix up and down , ice bath for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

(5) Add 15 ml of chloroform solution to the supernatant in step (4), mix well and ice bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH5.2) and 2.5 volumes of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 1 hours to precipitate the RNA, and centrifuge at 12000×g for 15 minutes at 4°C to obtain the precipitate;

(7) Wash the precipitation of step (6) with 75% ethanol for 3 times, and then place it at room temperature for 5-10 minutes to dry it, so as to obtain the pure RNA of the present invention.

Applying this embodiment, the total amount of cotton root RNA obtained is 1650 micrograms, and the yield is 550 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.77, which meets the needs of molecular biology experiments.

Example 8: Extract RNA samples from cotton roots stored in lysate for a long time

(1) Take 1 gram of fresh cotton root, wash it with water and grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 10 ml of pre-cooled RNA extraction buffer (pH6.5 ) and 200 microliters of β-mercaptoethanol, upside down to mix evenly, it was quickly frozen with liquid nitrogen and then stored in a -73°C refrigerator for 15 days;

(2) Thaw the frozen material mixture on ice, add 1 ml of sodium acetate, add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, 4 ° C, Centrifuge at 12000×g for 15 minutes, and recover the supernatant;

(3) Take the supernatant into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, place it in a -20°C refrigerator for 30 minutes, and centrifuge at 10,000×g for 15 minutes at 4°C;

(4) Discard the supernatant, dissolve the precipitate completely with 2ml RNA extraction buffer, then add 2ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 15 minutes, 4°C, 12000× Centrifuge at g for 15 minutes, and recover the supernatant;

(5) Add 2 ml of chloroform solution to the supernatant in step (4), mix well and ice bath for 15 minutes, centrifuge at 12000×g at 4°C for 15 minutes, and recover the supernatant;

(6) Add 1/10 volume of sodium acetate (pH 5.0) and 2.5 times volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 0.5 hours To precipitate RNA, centrifuge at 10,000×g for 15 minutes at 4°C to obtain a precipitate;

(7) washing with 75% ethanol to wash the precipitation of step (6) twice, and then place it at room temperature for 5-10 minutes to dry it, so that the pure RNA of the present invention can be obtained.

Applying this embodiment, the total amount of RNA obtained was 370 micrograms, and the yield was 370 micrograms/gram

Quality inspection: RNA remains intact through electrophoresis detection, and its A _260/280 = 1.82, meeting the needs of molecular biology experiments.

Claims (3)

1. A method for extracting RNA from cotton tissue, characterized in that it comprises the following steps: (1) After 1 weight part of cotton tissue is washed with water or directly ground into powder in liquid nitrogen or glass homogenizer, the tissue material is mixed with pre-cooled RNA in a volume/weight ratio of 1:5-15 Mix the extraction buffer and add 0-0.2 parts of β-mercaptoethanol and 0.8-1.2 parts of sodium acetate upside down to mix evenly, and place it on ice to obtain a sample mixture; (2) Add an equal volume of chloroform to the mixed solution obtained in step (1), turn it upside down to mix into one phase, place it on ice for 10-15 minutes, then centrifuge at 4°C for 15 minutes, and recover the supernatant; (3) Take the supernatant of step (2), add an equal volume of isopropanol at -20°C, mix up and down, place at -20°C for 0.5-1 hour to precipitate RNA, and centrifuge at 4°C for 15 minutes get sediment; (4) Dissolve the precipitate obtained in step (3) thoroughly with 1-5 parts by volume of RNA extraction buffer, then add 1-5 parts by volume of acid phenol solution, mix upside down, and ice bath for 10-15 minutes, Centrifuge at 4°C for 15 minutes, and recover the supernatant; (5) Add 1-5 parts by volume of chloroform solution to the supernatant in step (4), mix well and ice-bath for 10-15 minutes, centrifuge at 4°C for 15 minutes, and recover the supernatant; (6) Add 1/10 volume of sodium acetate and 2.5-3 times the volume of absolute ethanol to the supernatant of step (5), mix well, and place at -20°C for 0.5-1 hour to precipitate RNA, 4°C Centrifuge for 15 minutes to obtain the precipitate; (7) Wash the precipitate of step (6) 2-3 times with 75% ethanol solution, and then place it at room temperature for 5-10 minutes to dry it to obtain pure RNA; Wherein: the RNA extraction buffer is to use 472.64 grams of guanidine thiocyanate, 7.352 grams of sodium citrate, 5-10 grams of sodium lauryl sarcosine, 0.5-20 grams of polyvinylpyrrolidone 4000 ( PVP4000) was prepared by adding water to 950 ml, adjusting the pH to 6.0-6.5 with glacial acetic acid, and then adding water to make the volume to one liter; The sodium acetate solution is prepared by dissolving 408 grams of sodium acetate in water, adjusting the pH to 5.0-5.2 with glacial acetic acid and adding water to set the volume to one liter, and then autoclaving to obtain; The centrifugation is 9000×g-12000×g; Described chloroform solution is that the chloroform of 24 volume parts and the isoamyl alcohol of 1 volume part mix; The acid phenol solution is formed by mixing 1 volume part of water-saturated phenol (pH4.7-5.2) and 1 volume part of the above-mentioned chloroform solution; The 75% ethanol is mixed with 3 parts of absolute ethanol and 1 part of water; The water is pure water treated with 0.1% DEPC and sterilized by high pressure. 2. The method for extracting RNA from cotton tissue according to claim 1, wherein the cotton tissue is selected from cotton roots, hypocotyls, leaves, seedlings, flowers, calluses and fibers. 3. Application of the method for extracting RNA from cotton tissue according to claim 1 in cotton molecular biology.

Images