CN110643602A - A kind of extraction method of Huangshan magnolia root total RNA - Google Patents

Abstract

The invention provides a method for extracting total RNA from the roots of Magnolia japonica. The root tissue of Magnolia japonica is ground into powder in liquid nitrogen, mixed with an extraction solvent, and incubated to obtain an incubation mixture; extraction is performed to obtain an extraction supernatant Mix the extraction supernatant with lithium chloride solution, stand overnight and centrifuge to obtain total RNA precipitate; dissolve the total RNA precipitate in SSTE buffer, and then extract in chloroform/isoamyl alcohol mixture The supernatant was obtained by centrifugation; then mixed with absolute ethanol, left to stand at -70°C, and centrifuged to obtain a precipitate; the precipitate was washed with an ethanol solution to obtain a clean precipitate; Total RNA from the roots of Magnolia japonica was obtained from the ultrapure water treated with DEPC. Compared with the prior art, the present invention can successfully extract the total RNA from the roots of Magnolia japonica, with excellent purity and high extraction rate.

Description

A kind of extraction method of Huangshan magnolia root total RNA

technical field

The invention relates to a method for extracting RNA, in particular to a method for extracting total RNA from roots of Magnolia japonica.

Background technique

Magnolia cylindrica is a deciduous tree of the Magnolia family (Magnoliaceae) and is named after the type specimens were collected from Mount Huangshan. Huangshan magnolia has excellent material, beautiful tree shape, and its flowers have medicinal value. It is an important garden ornamental plant and an excellent aromatic plant. It is a precious national tertiary protection and endangered species. Therefore, it is of great significance to explore the Huangshan magnolia germplasm resources at the molecular level.

Huangshan magnolia is a perennial woody plant rich in secondary metabolites such as protein, polysaccharides and polyphenols. However, polysaccharides are easily co-precipitated with RNA, and polyphenols are easily oxidized to form quinones that bind irreversibly to RNA. These secondary metabolites cause great interference to RNA extraction. High-quality RNA extraction is a necessary prerequisite for molecular biology experiments, such as the construction of cDNA libraries, and the detection of Northern hybridization by fluorescence quantitative PCR. Therefore, it is of great significance to explore high-quality RNA extraction methods for Magnolia japonica.

Existing commonly used RNA extraction techniques include CTAB method, Trizol method, and many RNA extraction kits on the market, but these methods often have some problems such as poor timeliness. In addition, although many reports indicate that the above method can extract the RNA of many plants. However, due to the vast differences in material composition between different plants or different tissues of the same plant, the above method is not suitable for all materials. For some plant materials, it is necessary to gradually improve the ideal extraction method in the exploration.

For example, the "Research on the RNA Extraction Method of Medicinal Plant Purslane" published by Zhao Dan et al. uses the improved Trizol method to extract the medicinal plant Purslane RNA. The effect is remarkable (Zhao Dan, Chang Zhengyao, Hao Gangping, et al. Study on the extraction method of RNA from the plant purslane [J]. Shizhen Guoyi Guoyao, 2013, 24(1): 83-85.). But using this method to extract the root RNA of Magnolia japonica was not successful. From the appearance of the extracted RNA sample, it is milky white and viscous. Dilute the RNA precipitate with DEPC water, and it can be clearly found that there are obvious impurities that are insoluble in water at the bottom of the EP tube. This method can not meet the needs of related genetic engineering experiments of Magnolia japonica.

Therefore, it is necessary to provide a more targeted method for extracting high-quality root RNA of Magnolia japonica.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for extracting the total RNA of the roots of Magnolia japonica, through which high-purity total RNA of the roots of Magnolia japonica can be extracted, and the extraction rate is high and the cost is low.

The concrete technical scheme of the present invention is as follows:

A method for extracting total RNA from the roots of Magnolia japonica, comprising the following steps:

1), grind the fresh lateral root of Magnolia japonica into powder in liquid nitrogen, then mix the obtained root system powder of Magnolia japonica with an extraction solvent, and incubate to obtain an incubation mixture;

2), adding chloroform-isoamyl alcohol mixed solution to the incubation mixed solution obtained in step 1), extracting and centrifuging to obtain an extraction supernatant;

3), the extraction supernatant obtained in step 2) is mixed with lithium chloride solution, left standing overnight, and centrifuged to obtain total RNA precipitation;

4), adding ethanol solution to the total RNA precipitate obtained in step 3), rinsing and centrifuging to obtain a precipitate;

5), dissolve the precipitate obtained in step 4) in SSTE buffer, then mix with chloroform-isoamyl alcohol mixed solution, extract and centrifuge to obtain centrifugal supernatant;

6), mixing the centrifugal supernatant obtained in step 5) with absolute ethanol, standing, and centrifuging to obtain a precipitate;

7), mixing the precipitate obtained in step 6) with an ethanol solution, rinsing and centrifuging to obtain a precipitate, adding absolute ethanol to the obtained precipitate, rinsing and centrifuging to obtain a precipitate; namely, the total RNA of the roots of Magnolia japonica.

The yellow mountain magnolia described in step 1) is the biennial yellow mountain magnolia cultivated in the greenhouse;

Further, in step 1), the freshly developed lateral roots of Magnolia japonica were selected, cut with scissors, rinsed in running water, placed in liquid nitrogen, and stored at -80°C for RNA extraction.

In step 1), grind until any granular root system is invisible to the naked eye.

The extraction solvent in step 1) contains CTAB extract and β-mercaptoethanol; the volume ratio of the CTAB extract to β-mercaptoethanol is 100:2; the CTAB extract is under high pressure at 121°C Sterilize for 20 min, then add β-mercaptoethanol.

Preferably, the CTAB extract contains 2% CTAB cetyltrimethylammonium bromide, 2% PVP polyvinylpyrrolidone, 0.1 mol/L pH=8.0 Tris-HCl buffer solution, 2 mol/L NaCl, 25mmol/L EDTA-Na ₂ (pH8.0) and DEPC-treated ultrapure water.

The specific configuration method of the CTAB extract is as follows:

Dissolve 2.4g Tris in 100ml 1/1000 DEPC water, add hydrochloric acid to pH=8, dissolve 1.86g EDTA-Na ₂ ·2H ₂ O in 100ml 1/1000 DEPC water, add hydrochloric acid to pH=8, two After mixing, add 4g CTAB, 4gPVP and 23.4gNaCl.

In step 1), the temperature of the extraction solvent is 65°C; in step 1), the mixing refers to vortex mixing, and the vortex mixing time is not less than 30s;

In step 1), the incubation conditions are as follows: the incubation temperature is 65° C. and the incubation time is 3-5 min; during the incubation period, the vortex is vortexed 3-5 times, and each vortex is 10 s.

Preferably, in step 1), the ratio of the volume of the extraction solvent to the mass of the root powder of Magnolia japonica is: 6 μL: 1.5-2 mg. Preferably, each 600 μL of the extraction solvent is mixed with 150-200 mg of Magnolia japonica root powder. In the present invention, it is considered to ensure that the sample tissue is evenly and thoroughly dispersed in the extracting solution, so the plant sample material cannot be too much, otherwise the sample tissue will be lumpy. Combining various factors, it was found that 150-200 mg of sample material mixed with 600 μL of CTAB extract is the most suitable.

Step 1) The mortar and medicine spoon used in the grinding process should be pre-cooled with liquid nitrogen before use. Liquid nitrogen should be added at any time during the grinding process to ensure that the powder is not thawed before adding to the extraction solution, otherwise RNA degradation may occur.

The CTAB extract of the present invention contains PVP polyvinylpyrrolidone, and the high-quality concentration of PVP can effectively remove polysaccharides and polyphenols in plant sample tissues, and can also remove some other pigment substances and lipids, because the plant tissue of Magnolia japonica contains oil cells, Polysaccharides, polyphenols and other substances, therefore, the CTAB extract of the present invention uses a high concentration of PVP to remove lipids, polysaccharides, polyphenols and other substances in the sample tissue. In addition, most cells can only perform activities in a very narrow pH range, so maintaining a normal pH environment is not easy for cells to be inactivated. The present invention uses 0.1mol/L Tris-HCl buffer solution (pH8.0) to provide a buffer environment to prevent Nucleic acid is destroyed, which is conducive to the stability of RNA, making it difficult to degrade. In the present invention, CTAB is used as a detergent, can dissolve cell membrane, can form complex with nucleic acid, and is soluble in high salt solution. Therefore, when the salt concentration of the solution is reduced to a certain extent (such as 2mol/L NaCl), the CTAB-nucleic acid complex can be separated from the protein and polysaccharide substances by precipitation from the solution and centrifugation. The pH of inhibiting RNase is 8.0, and 25mmol L EDTA-Na ₂ (pH8.0) of the present invention provides an optimal environment for inhibiting RNase activity.

In the chloroform-isoamyl alcohol mixed solution described in step 2), the volume ratio of chloroform and isoamyl alcohol is 24:1;

Preferably, the mixing volume ratio of the incubation mixed solution and the chloroform-isoamyl alcohol mixed solution described in step 2) is 1:1;

More preferably, 600 μL of chloroform-isoamyl alcohol mixed solution is used for every 600 μL of the incubation mixture; it is found that adding 600 μL of extracting solution is the most suitable.

Step 2) is specifically as follows: adding chloroform-isoamyl alcohol mixed solution to the incubation mixed solution obtained in step 1), vortex mixing, and then centrifuging at 10000 r/min at room temperature for 15 min, and taking the supernatant, that is, The supernatant must be extracted.

Further, the vortex mixing in step 2) is as follows: under room temperature conditions, the vortex time is 1 min.

Preferably, the extraction supernatant obtained in step 2) is processed again by the same extraction and centrifugation method as in step 2).

The invention performs vortex mixing to ensure that the plant sample tissue is completely and evenly suspended in the solution, ensures that the sample tissue is evenly dispersed, prevents the material from being lumpy, and improves the extraction rate and purity. In addition, when the temperature of CTAB is lower than 15 °C, CTAB will be precipitated, and the nucleoprotein cannot be depolymerized, and the nucleic acid will be released. Therefore, vortex for 1 min at room temperature to fully mix the CTAB extract with the plant sample tissue, thereby separating the RNA.

In step 3), the volume ratio of the extraction supernatant to the lithium chloride solution is 3:1; the concentration of the lithium chloride solution is 8 mol/L.

Preferably, in step 3), the condition of standing overnight: standing at 3-5° C. for 8-16 h;

In step 3), the centrifugation treatment is as follows: 10000 r/min under the condition of 4°C, centrifugation for 20 min.

In step 4), the ethanol solution is an ethanol solution with a volume concentration of 70%, and is configured by using RNase-free DEPC-treated water with ultrapure water.

In step 4), the rinsing and centrifugation refers to centrifugation at 10,000 r/min for 5 min at 4°C.

In step 5), the amount of SSTE buffer is 500 μL relative to the precipitate prepared per 600 μL of the incubation mixture;

Preferably, the SSTE buffer is composed of: 0.5% SDS sodium dodecyl sulfate, 1.0mol/L NaCl, 10mmol/L Tris-HCl (pH=8.0) and 1mmol/L EDTA-Na ₂ (pH=8.0) .

The specific configuration method of the SSTE buffer is as follows: 0.12g Tris is dissolved in 50ml 1/1000 DEPC water, then hydrochloric acid is added to pH=8, 0.037g EDTA-Na ₂ ·2H ₂ O is dissolved in 50 ml 1/1000 DEPC water, and then Hydrochloric acid was added to pH=8, and 0.5g SDS and 5.84g NaCl were added after mixing the two, and then autoclaved at 121°C for 20min.

The high concentration of SDS in the SSTE buffer of the present invention can destroy the cell membrane, thereby rapidly inhibiting the RNase in the cell and ensuring the integrity of the RNA. Tris-Cl can destroy the combination of protein and nucleic acid and release the nucleic acid, while EDTA can effectively Inhibits RNase activity and prevents RNA degradation, and the action of NaCL increases salt concentration and prevents RNA degradation.

In step 5), the volume ratio of SSTE buffer and chloroform-isoamyl alcohol mixed solution is 1:1;

Specifically in step 5):

The precipitate obtained in step 4) was dissolved in the SSTE buffer, then vortexed with an equal volume of chloroform-isoamyl alcohol mixture at room temperature for 1 min, and then centrifuged at 10,000 r/min at 4°C for 15 min, and the supernatant was taken;

In step 6), the volume ratio of the centrifugal supernatant to absolute ethanol is 1:2.

In step 6), the conditions of standing are: the standing time is 30 minutes under the condition of -70°C, or the standing time is 2 hours under the condition of -20°C.

The centrifugation in step 6) refers to: centrifugation at 10000 r/min at 4°C for 20 min.

Compared with DNA, RNA is easily degraded, and the content of RNA extracted from samples of the same quality is lower, so it is necessary to prolong the precipitation time to ensure complete precipitation of RNA. And RNA has low solubility in cold absolute ethanol, while protein solubility is high. RNA is more likely to precipitate at ultra-low temperature.

In step 7), the amount of the ethanol solution is 400 μL relative to the precipitate obtained after 600 μL of the incubation mixture is processed in steps 2) to 6).

Step 7) is specifically as follows: using an ethanol solution for the precipitate obtained in step 6), centrifuging for 5 min at 10000 r/min at 4 °C, to obtain a precipitate, and adding absolute ethanol to the obtained precipitate, at 10000 r/min at 4 °C, Centrifuge for 5 min to obtain a precipitate.

The precipitate obtained in step 7) was placed openly, added with DEPC-treated ultrapure water, and stored.

Under the described open placement conditions: placed at room temperature for 2-3min;

The mass ratio of sediment to DEPC-treated ultrapure water was 3.7-9.6:10-20.

All the above steps are performed in a clean bench.

The present invention successfully extracts total RNA from the root material of Magnolia japonica through the synergistic effect of the above steps; and the extraction method is simple and easy to implement, thereby meeting the requirements of subsequent molecular biology experiments and related research. At the same time, the extraction rate of this extraction method is high.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention.

Fig. 1 is the agarose gel electrophoresis figure that the method of the present invention extracts RNA;

Fig. 2 is the agarose gel electrophoresis figure of RNA extracted by Trizol method;

Figure 3 shows the detection of amplified products by agarose gel electrophoresis.

Detailed ways

The specific embodiments of the present invention will be described in detail below, and it should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

The reagents and equipment used in the present invention and the precautions for operation are as follows:

1) The CTAB extract was prepared with ultrapure water treated with CTAB, PVP, Tris, NaCl, EDTA-Na ₂ 2H ₂ O and DEPC. The reagents required for the above configuration were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; CTAB Before use, the extract should be checked for precipitation. If there is precipitation, it should be dissolved at 65°C and used again; the lithium chloride solution is prepared with DEPC-treated ultrapure water, and lithium chloride is purchased from Shenggong Bioengineering ( Shanghai) Co., Ltd.; chloroform-isoamyl alcohol mixed solution (24:1, v/v) solution was prepared using chloroform and isoamyl alcohol; chloroform was purchased from Suzhou Kelun Chemical Co., Ltd., and isoamyl alcohol was purchased from Tianjin Kai Tong Chemical Reagent Co., Ltd.; RNase-free (RNase-free) 1.5 ml centrifuge tubes were purchased from Corning Corning Company, and RNase-free 10-1000 μL tips used in the present invention were purchased from Hefei Zhihong Tech Biotechnology Co., Ltd. The vortex instrument used in the vortex of the present invention is a QL-861 vortex mixer; the ethanol solution uses an ultrapure water configuration of RNase-free DEPC-treated water.

2) The preparation method of the DEPC-treated ultrapure water used in the present invention: add DEPC diethyl pyrocarbonate to the ultrapure water, leave it for 12 hours overnight, and autoclave at 121° C. for 20 minutes to remove DEPC to obtain RNase-free DEPC treated water of ultrapure water. Among them, the volume ratio of ultrapure water and DEPC is 1000:1. Unsterilized DEPC-treated water and DEPC reagents are prohibited from being used in experiments, and sterilized DEPC-treated water can no longer be used to treat tips and other items to avoid contamination.

3) The devices such as reagents and pipette tips used in the present invention are all RNase-free, and the specific processing steps are as follows: soak the pipette tips, centrifuge tubes, etc. in ultrapure water treated with DEPC overnight, take out and autoclave to remove DEPC, which is RNase -free, can be used for RNA experiments.

4) The mortars and medicine spoons used in the experiment were soaked in ultrapure water with DEPC overnight before use, taken out for autoclaving, and dried to remove RNase. The mortar and spoon should be pre-cooled with liquid nitrogen before use. Liquid nitrogen should be added at any time during the grinding process to ensure that the powder is not thawed before adding to the extraction solution, otherwise RNA degradation may occur.

5) Wipe the ultra-clean workbench with 75% ethanol and absorbent cotton before the operation process of the present invention, place the required pipettes, suction tips, mortars, medicine spoons, etc. on the table, light the alcohol lamp, and turn on the ultra-clean work. Use it after UV disinfection for 30 minutes; keep the alcohol lamp on during work.

6) During the implementation of the present invention, gloves should be changed frequently, and gloves should be changed when they come into contact with surfaces such as door handles, refrigerator doors, laboratory countertops, table and chair surfaces, etc.; Doors and windows, reduce talking, etc. to reduce the chance of RNase (RNase) contamination.

Example 1

A method for extracting total RNA from the roots of Magnolia japonica, comprising the following steps:

1) Dissolve 2.4g Tris in 100ml 1/1000 DEPC water, add hydrochloric acid to adjust to pH=8 to obtain a Tris-HCl buffer solution; dissolve 1.86g EDTA-Na ₂ ·2H ₂ O in 100 ml 1/1000 DEPC water , add hydrochloric acid to adjust to pH=8 to obtain EDTA-Na ₂ solution, mix the two solutions prepared above, add 4g CTAB, 4g PVP and 23.4g NaCl, mix well to obtain CTAB extract; extract the obtained CTAB The solution was autoclaved at 121 °C for 20 min, then 2% β-mercaptoethanol by volume of CTAB extract was added and mixed with CTAB extract as an extraction solvent, and placed in a 65 °C water bath to preheat; 0.5 g of fresh lateral root material of biennial Magnolia japonica cultivated in the greenhouse was placed in a mortar pre-cooled with liquid nitrogen, and ground fully with liquid nitrogen until no granular root system was visible to the naked eye, and became powder. The mortar was allowed to stand for 2 minutes at room temperature (to prevent CTAB from being crystallized because the mortar was too cold); at room temperature, add 1.5 mL of the above-mentioned extraction solvent preheated to 65°C and grind in the mortar to mix the extraction solvent and powder evenly. Obtain a mixture of extraction solvent and powder; take 600 μL of the above mixture into a 1.5 mL centrifuge tube, immediately use a vortex mixer to mix for 30 s, and finally place it in a 65°C water bath for 5 min, and take it out every one minute during the incubation period. , and vortexed at room temperature for 10 s each time to obtain the incubation mixture.

2) Take 600 μL of the incubation mixture prepared in step 1) into a centrifuge tube, shake well, cool to room temperature, and add 600 μL of chloroform-isoamyl alcohol with a volume ratio of 24:1 chloroform and isoamyl alcohol to the centrifuge tube. The solution was mixed by vortexing at room temperature for 1 min, and then centrifuged at 10,000 r/min for 15 min at room temperature; then the supernatant was aspirated; the obtained supernatant was placed in a centrifuge tube, and an equal volume of chloroform and isopropyl alcohol were added to the centrifuge tube at room temperature. A chloroform-isoamyl alcohol mixed solution with a volume ratio of amyl alcohol of 24:1 was mixed by vortexing at room temperature for 1 min, and then centrifuged at 10,000 r/min for 15 min at room temperature; , When sucking the supernatant, in order to reduce the contamination of impurities, you should avoid sucking the lower layer of solute, and try not to suck the liquid that is in contact with the second layer at the bottom of the uppermost layer.

3) Add the extraction supernatant obtained in step 2) into another new 1.5ml centrifuge tube, and then add 8 mol/L LiCl solution that is 1/3 of the volume of the extraction supernatant, and invert it upside down to make it Mix well; then place at 4°C overnight for 14h to precipitate; finally, centrifuge the centrifuge tube at 10,000 rpm for 20min at 4°C, take the precipitate, and obtain the crudely extracted total RNA precipitate. Among them, in order to prevent incomplete precipitation of RNA, the supernatant after centrifugation should be transferred to a new RNase-free centrifuge tube, and the supernatant should be discarded after centrifugation to ensure complete precipitation of RNA.

4) Rinse and precipitate the crudely extracted total RNA precipitate obtained in step 3) with 50 μL of 70% ethanol solution by volume, centrifuge at 10,000 r/min for 5 min at 4° C., discard the supernatant, and dry the precipitate at room temperature for 30 s;

5) Place the dried precipitate in step 4) in 500 μL SSTE buffer, and after mixing, add an equal volume of chloroform-isoamyl alcohol with a volume ratio of 24:1 chloroform-isoamyl alcohol mixed solution, vortex at room temperature Spin for 1 min, then centrifuge at 10,000 r/min at 4°C for 15 min, and take the supernatant;

The specific configuration method of the SSTE buffer is as follows: 0.12g Tris is dissolved in 50ml 1/1000 DEPC water, then hydrochloric acid is added to pH=8, 0.037g EDTA-Na ₂ ·2H ₂ O is dissolved in 50 ml 1/1000 DEPC water, and then Hydrochloric acid was added to pH=8, and 0.5g SDS and 5.84g NaCl were added after mixing the two, and then autoclaved at 121°C for 20min.

6) Transfer the supernatant obtained in step 5) to a new 1.5ml centrifuge tube, add 2 times the volume of absolute ethanol of the supernatant, and after mixing, let stand at -70°C for 30min; then Centrifuge at 10,000 rpm for 20 min at 4°C, discard the supernatant, and obtain a precipitate.

7) Dissolve the precipitate prepared in step 6) in 400 μL of 70% ethanol solution by volume, then centrifuge at 10,000 rpm for 5 min at 4°C, carefully pour out the supernatant to obtain a precipitate, and re-suspend the obtained precipitate. Add 400 μL of anhydrous ethanol solution, centrifuge at 10,000 rpm for 5 min at 4°C, and aspirate the supernatant with a pipette to obtain a precipitate.

8) Open the cap of the centrifuge tube obtained in step 7), and place it in an ultra-clean bench to dry at room temperature for 2 minutes; after the ethanol is completely evaporated, total RNA from the roots of Magnolia japonica is obtained.

9) Dissolve the total RNA precipitate of Magnolia japonica root system prepared in step 8) with 20 μL of DEPC-treated ultrapure water to obtain a total RNA sample 1 from the root of Magnolia japonicae, and store at -80°C.

Example 2

A method for extracting total RNA from the roots of Magnolia japonica, except that LiCl solution was added in step 3) in the extraction process of this embodiment, and then placed at 4° C. overnight for 16 hours; other things were the same as in Example 1, and total RNA from the roots of Magnolia japonica was finally obtained Sample 2.

Example 3

A method for extracting total RNA from the roots of Magnolia japonica. In the extraction process of this embodiment, LiCl solution is added in step 3), and then placed at 4° C. for 12 hours overnight; other things are the same as in Example 1, and a sample of total RNA from the roots of Magnolia japonica is finally obtained. 3.

Comparative Example 1

A method for extracting total RNA of Magnolia japonica root system (Trizol method), comprising the following steps:

1) Take 0.3 g of fresh lateral roots of 2-year-old Magnolia japonica cultivated at room temperature, add 1% polyvinylpyrrolidone (PVP) of the sample volume and grind into powder in liquid nitrogen, take 200 mg of the powder in a 1.5 mL centrifuge tube pre-cooled in liquid nitrogen , add 1 mL of Trizol reagent and 10 μL of β-mercaptoethanol with a mass-to-volume ratio of 1%, let stand for 10 min at room temperature to fully lyse, then centrifuge at 4°C for 5 min at 12000 r/min, transfer the supernatant to a new centrifuge tube ;

2) Add 200 μL of 5mol/L NaCl solution to the supernatant obtained in step 1), add 200 μL of chloroform after shaking, shake and mix evenly, let stand for 15 minutes at room temperature, centrifuge at 12000 r/min for 15 minutes at 4°C, and draw the supernatant phase into a new centrifuge tube;

3) adding an equal volume of chloroform to the clear liquid obtained in step 2) for extraction, centrifuging at 12000 r/min at 4°C for 15 min, and sucking the upper aqueous phase into a new centrifuge tube;

4) Add equal volume of phenol-chloroform-isoamyl alcohol mixture to the supernatant obtained in step 3), mix well, let stand for 5 min at room temperature, centrifuge at 12000 r/min for 15 min at 4°C, and take the supernatant to another fresh in a centrifuge tube; the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1.

5) Add 0.5 mL of isopropanol to the supernatant obtained in step 4), mix well, place at room temperature for 5-10 min, centrifuge at 12000 r/min for 10 min at 4°C, discard the supernatant, and obtain RNA precipitation;

6) Add 1 mL of 75% ethanol solution by volume to step 5) RNA precipitation, centrifuge at 8000 r/min at 4°C for 5 min, discard the supernatant, and dry at room temperature;

7) Add 20 μL of DEPC-treated ultrapure water to the precipitate after drying in step 6) to dissolve the clean precipitate to obtain total RNA sample 4 from the root of Magnolia japonica; store at -80°C.

Comparative Example 2

A method for extracting total RNA from the roots of Magnolia japonica (Trizol method), adding an equal volume of phenol-chloroform-isoamyl alcohol mixture in step 4) in the extraction process of this comparative example to extract twice, and the others are the same as in Comparative Example 1, and the final preparation is made. The total RNA sample 5 from the root of Magnolia japonica was obtained.

Detection:

The total RNA samples 1-5 of the roots of Magnolia japonica were detected by TECAN microplate reader, and the ultrapure water treated with 2 μL DEPC was used as a blank, and 2 μL RNA samples were drawn for detection. The specific test sample indicators are shown in Table 1.

Table 1

TECAN microplate reader detects RNA indicators extracted from samples 1-5

Material Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 RNA concentration (μg/μL) 303.02 476.65 187.04 -0.08 -0.56 OD₂₆₀/OD₂₈₀ 2.23 2.25 2.22 0.33 1.27 OD₂₆₀/OD₂₃₀ 2.47 2.48 2.40 0.50 0.54

The total RNA samples 1-5 from the roots of Magnolia japonica were detected by agarose gel electrophoresis. The detection conditions were: 1% agarose gel, voltage 108V, current 100mA, time 30min; the detection results are shown in Figure 1 and Figure 2 (marker is the molecular weight mark, the band size from bottom to top is: 100, 250, 500, 750, 1000, 1500 and 2000 bp), Figure 1 is the present invention, Figure 2 is the Trizol method. It can be seen from Figure 1 that the electrophoretic bands of total RNA extracted from the roots of Magnolia japonica by this method are clear and have good integrity, which can meet the needs of downstream experiments. In Figure 1, the 28s and 18s bands can be clearly seen, while in Figure 2 there is no band and there is a stray band tail.

Reverse transcription PCR experiment:

1. Reverse transcription: use Tiangen "FastQuant cDNA First Strand Synthesis Kit" for reverse transcription. 50ng-2μg total RNA can create a 20μL reaction system:

(1) Thaw the template RNA on ice, 5×gDNA Buffer, FQ-RT Primer Mix, 10×Fast RTBuffer, RNase-Free ddH ₂ O thaw at room temperature, and quickly place on ice after thawing. Vortex and mix each solution before use, and centrifuge to collect the liquid remaining on the tube wall;

The following operation steps are carried out on ice. In order to ensure the accuracy of the preparation of the reaction solution, each reaction should be prepared into Mix first, and then dispensed into each reaction tube;

(2) Prepare a mixed solution according to the genomic DNA removal system in Table 2, mix well, centrifuge, and place at 42° C., incubate for 3 min, and then place on ice.

Table 2 gDNA removal reaction system

Composition Usage amount 5×g DNA Buffer 2μL Total RNA 5μL RNase-Free ddH2O Supplement to 10 μL

(3) Prepare the mixed solution according to the reverse transcription reaction system of Table 3

Table 3 Reverse transcription reaction system

Composition Usage amount 10×Fast RT Buffer 2μL RT Enzyme Mix 1μL FQ-RT Primer Mix 2μL RNase-Free ddH₂O Supplement to 10 μL

(4) Add the Mix in the reverse transcription reaction to the reaction solution of the gDNA removal step, and mix thoroughly.

(5) 42℃, incubate for 15min.

(6) Incubate at 95°C for 3 min and place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

reverse transcription PCR:

Actin primers were designed from the transcriptome gene of Magnolia japonica,

Upstream primer F: 5'-CGAGTTGTACGTGGTCTC-3',

Primer downstream R: 5'-CATCCTGTCCTCCTTACTG-3';

Amplified sequence length: 543bp, cDNA diluted 10 times, primer 10μM. PCR system: 1 μL of cDNA obtained by reverse transcription of the RNA extracted in Example 1; 1.25 μL of upstream primer; 1.25 μL of downstream primer; 12.5 μL of 2×FastTaq Premix (purchased from Tolo Harbour Biotechnology Co., Ltd.) Ultrapure water was added to 25 μl. Reaction conditions: denaturation at 94 °C for 3 min; melting at 94 °C for 30 s, annealing at 52 °C for 1 min, extension at 72 °C for 1 min and 30 s for 35 cycles; extension at 72 °C for 10 min.

The amplification product 1-amplification product 5 obtained from the cDNA obtained by reverse transcription of the RNA extracted in Example 1 was detected by agarose gel electrophoresis, and the detection conditions were: 1% agarose gel, voltage 108V, current 100mA, time 30min, the test results are shown in Figure 3.

SEQUENCE LISTING

<110> Anhui Normal University

<120> A kind of extraction method of Huangshan magnolia root total RNA

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

<213> Upstream primer

<400> 1

5'-cgagttgtac gtggtctc-3' 18

<210> 2

<211> 19

<212> DNA

<213> Downstream Primer R

<400> 2

5'-catcctgtcc tccttactg-3' 19

Claims (10)

1. an extraction method of Magnolia japonica root total RNA, is characterized in that, described extraction method may further comprise the steps: 1), grind the fresh lateral root of Magnolia japonica into powder in liquid nitrogen, then mix the obtained root system powder of Magnolia japonica with an extraction solvent, and incubate to obtain an incubation mixture; 2), adding chloroform-isoamyl alcohol mixed solution to the incubation mixed solution obtained in step 1), extracting and centrifuging to obtain an extraction supernatant; 3), the extraction supernatant obtained in step 2) is mixed with lithium chloride solution, left standing overnight, and centrifuged to obtain total RNA precipitation; 4), adding ethanol solution to the total RNA precipitate obtained in step 3), rinsing and centrifuging to obtain a precipitate; 5), dissolve the precipitate obtained in step 4) in SSTE buffer, then mix with chloroform-isoamyl alcohol mixed solution, extract and centrifuge to obtain centrifugal supernatant; 6), mixing the centrifugal supernatant obtained in step 5) with absolute ethanol, standing, and centrifuging to obtain a precipitate; 7), mixing the precipitate obtained in step 6) with an ethanol solution, rinsing and centrifuging to obtain a precipitate, adding absolute ethanol to the obtained precipitate, rinsing and centrifuging to obtain a precipitate; namely, the total RNA of the roots of Magnolia japonica. 2 . The extraction method according to claim 1 , wherein in step 1), the mixing refers to vortex mixing, and the vortex mixing time is not less than 30s. 3 . 3. The extraction method according to claim 1 or 2, characterized in that, in step 1), the incubation conditions are: the incubation temperature is 65°C, and the incubation time is 3-5min; vortex 3-5 times during incubation , vortex for 10s each time. 4. The extraction method according to claim 1, wherein step 2) is specifically: adding chloroform-isoamyl alcohol mixed solution to the incubation mixed solution obtained in step 1), then vortex mixing, and then centrifuging , 10000r/min at room temperature, centrifuge for 15min, take the supernatant, that is, extract the supernatant. 5. The extraction method according to claim 1, wherein the ethanol solution is an ethanol solution with a volume concentration of 70%, and is configured using the ultrapure water of RNase-free DEPC-treated water. 6. extraction method according to claim 1, is characterized in that, in step 5), described SSTE buffer solution is composed: 0.5%SDS sodium dodecyl sulfate, 1.0mol/L NaCl, 10mmol/L Tris-HCl (pH=8.0) and 1 mmol/LEDTA- _Na2 (pH=8.0). 7. extraction method according to claim 1 or 6, is characterized in that, in step 5), the volume ratio of SSTE buffer and chloroform-isoamyl alcohol mixed solution is 1:1. 8. The extraction method according to claim 1 or 6, wherein in step 6), the volume ratio of the centrifugal supernatant to absolute ethanol is 1:2. 9. The extraction method according to claim 1 or 6, wherein in step 6), the standing condition is: the standing time is 30min under the -70 ℃ condition, or, the standing still under the -20 ℃ condition Set time is 2h. 10. The extraction method according to claim 1 or 6, characterized in that, step 7) is specifically: using the precipitate obtained in step 6) with ethanol solution, 10000r/min under 4 DEG C of conditions, centrifuged for 5min to obtain the precipitate , the obtained precipitate was then added with absolute ethanol, and centrifuged at 10,000 r/min at 4°C for 5 min to obtain a precipitate.

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