CN116445471A - Extracting solution for extracting total RNA of southern medicine plants, application and extracting method thereof - Google Patents
Extracting solution for extracting total RNA of southern medicine plants, application and extracting method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of RNA extraction, in particular to an extracting solution for extracting total RNA of a southern herbal plant, application thereof and an extracting method thereof. The invention also relates to application of the extracting solution in extracting total RNA of southern medicine plants; and provides a method for extracting total RNA of the southern medicine plants. Compared with RNA extraction kits and other RNA extraction methods, the method can fully crack the nuclease, improve the yield and purity of RNA, has high extraction efficiency, is simpler to operate than other hand-held methods, has low reagent price, and effectively reduces economic cost.
Description
Technical Field
The invention relates to the technical field of RNA extraction, in particular to an extracting solution for extracting total RNA of a southern herbal plant, application thereof and an extracting method thereof.
Background
At present, along with the rapid development of modern biological technology research, RNA extraction and application become an essential experimental link of scientific research, but RNA is of a single-chain structure, unlike DNA, and does not form a double-helix structure, so that the structural stability is poor, and the RNA is extremely easy to degrade, thereby influencing the subsequent experimental research result.
The existing common plant RNA extraction methods include a CTAB method, an SDS method, a rapid SDS method, an improved thermal boric acid method and the like, and the methods are researched and reported on plants such as yew, lawn plants, tea plums, lily and the like, wherein the isothiocyanate can strongly inhibit the activities of RNase in tissues and extracting solutions, but the isothiocyanate method is only applicable to tissue materials with low secondary metabolite content, SDS has a strong cracking effect at normal temperature, and can effectively remove polysaccharide in the plant tissues by combining phenol extraction and chloroform extraction; in the thermal boric acid method, boric acid can form a compound with phenolic substances, so that the interference of polyphenol is avoided, and proteinase K and potassium acetate can remove protein and polysaccharide respectively, but the method has the advantages of more required medicines, high price and complex operation; CTAB can coprecipitate with protein, polyphenol and neutral polysaccharide in a high ion concentration solution, and simultaneously, nucleic acid is dissolved to achieve the purpose of separating nucleic acid, the CTAB method has high extraction efficiency and high purity of products, and the required medicines are simple, so that the method is an effective method for extracting total RNA of plant tissues rich in polysaccharide and polyphenol.
The existing methods for extracting total RNA from different tissues of southern medicinal plants such as patchouli are not much researched, and the existing biotechnology companies continuously research and develop RNA extraction kits, so that the kits are popularized. The kit has the advantages of simple steps, easy operation and good stability in RNA extraction, but also has the problems of general extraction efficiency, high price, difficult long-time preservation of reagents and the like. The traditional hand-held method is poor in effect, complex and complex in operation, more in reagent configuration steps, too much organic reagent residues exist in the extraction steps, the RNA extraction concentration is not high, and often many experimental results do not reach the expected effect.
Disclosure of Invention
The invention provides an extracting solution and an extracting method for extracting total RNA of a southern medicine plant, which aims to solve the problems that the operation is complicated and complicated, the reagent configuration steps are more, and the extraction concentration of RNA is not high and the extraction efficiency cannot reach the expected value due to the excessive organic reagent residues in the extraction steps. The extracting solution can improve the extracting yield and purity of RNA, and has high extracting efficiency, relatively simple operation and low cost.
In order to solve the technical problems, the invention adopts the following technical scheme: an extracting solution for extracting total RNA of southern medicine plants comprises a CTAB buffer solution and an SSTE solution, wherein the pH values of the CTAB buffer solution and the SSTE solution are 7.5-8.0,
the CTAB buffer solution comprises 20g/L PVP, 0.05mol/L tris, 0.0125mol/L EDTA, 0.055mol/L CTAB and 1mol/L NaCl;
the SSTE solution comprises 0.95mol/L NaCl, 0.02mol/L SDS, 0.01mol/L tris and 0.001mol/L EDTA;
the pH value of the tris is 7.5-8.0, and the pH value of the EDTA is 7.5-8.0.
Furthermore, the application of the extracting solution in extracting total RNA of the southern medicinal plants is also within the protection scope of the invention.
Also provided is a method for extracting total RNA of a southern medicine plant, which comprises the following steps:
s1: preparing a basic reagent and an extracting solution one day in advance, sterilizing, and preparing a gun head and a mortar for sterilization;
s2: quickly grinding the south medicinal plant leaves with liquid nitrogen, subpackaging into 2-tube samples after grinding, and quickly freezing the samples in the liquid nitrogen;
s3: adding CTAB buffer solution and beta-mercaptoethanol into a sample, uniformly mixing on a vortex instrument, and then heating in a water bath kettle;
s4: adding chloroform and isoamyl alcohol with the volume ratio of 24:1 into each tube of sample, uniformly mixing and centrifuging, and repeating for one time;
s5: sucking the supernatant obtained in the step S4, combining the 2-tube supernatant obtained in the step S4 into 1 tube, adding LiCl, uniformly mixing, and standing for 12-15h;
s6: centrifuging the sample placed in the step S5, discarding the supernatant, adding SSTE solution, chloroform and isoamyl alcohol with the volume ratio of 24:1, uniformly mixing and centrifuging;
s7: transferring the supernatant to a new centrifuge tube, adding absolute ethanol with the same volume, standing at-80 ℃ for 30min, and centrifuging;
s8: removing supernatant, adding 1ml of 75% ethanol to wash precipitate for 1-2 times, suspending the precipitate, and centrifuging at 4deg.C for 5min at 8000 r.min;
s9: discarding supernatant, air drying, adding DEPC treated water for dissolving, and preserving for standby;
s10: RNA quality and concentration were measured.
Thus, the CTAB extract of the present invention provides a suitable buffer environment to prevent the destruction of nucleic acids during cell lysis. RNA is easy to be catalyzed by alkali to hydrolyze, so SSTE buffer with pH value of 7.5-8.0 is used for dissolving RNA, so that DNA and protein can enter an organic phase in the extraction process, RNA enters an aqueous phase, and RNA with higher purity can be obtained by comparison. In the operation process, the RNA extraction concentration can be greatly improved by combining two samples into one tube, and the water bath time is shortened from 30min to 5min after beta-mercaptoethanol is added and vortex is carried out, and when chloroform is added: after isoamyl alcohol, the shaking time is prolonged to 1min from 30s, and meanwhile, the step of water bath is added, so that the extraction efficiency can be effectively improved. In the process, the chloroform added for the second time is removed by absolute ethanol: after isoamyl alcohol, the added organic reagent can be better washed away, so that the influence of excessive residues on the follow-up is prevented.
Further, in the step S3, the CTAB extracting solution is preheated at 65 ℃ in advance, 0.8-1ml of CTAB and 50-60 mu L of beta-mercaptoethanol are added into a centrifuge tube added with the sample, and the mixture is uniformly mixed in a vortex instrument and then is heated for 5min in a water-soluble pot at 65 ℃.
Further, in the step S4, 0.6-0.8mL of chloroform and isoamyl alcohol with the volume ratio of 24:1 are added into each tube, and the mixture is vigorously vibrated for 1min and water-bath is carried out for 10min; the operation is repeated for 1 time, without water bath, after direct shaking, the centrifuge is precooled at 4 ℃ and centrifuged for 10min at 12000r min < -1 >.
In the step S5, the 2-tube supernatant obtained in the step S4 is firstly combined into 1 tube, then 200-300 mu L of 12mol/L LiCl is added, and the mixture is uniformly mixed and placed in a refrigerator at 4 ℃ for 12-15h.
Further, in the step S6, 0.4-0.6ml of SSTE solution is added into the tube, and the SSTE solution is heated in a water bath kettle at 65 ℃ in advance.
Further, in the step S7, absolute ethyl alcohol in a centrifuge tube is placed in a refrigerator at the temperature of minus 20 ℃ for one day in advance, and is placed at the temperature of minus 80 ℃ for 30min, and then is placed at the temperature of 4 ℃ for 12000 r.min -1 Centrifuging for 20 min.
Further, in the step S9, the mixture is dried for 5min, 20-30 mu L of DEPC treated water is added for dissolution, and the mixture is placed on ice to be detected or directly stored at the temperature of minus 80 ℃.
Further, in S10, the quality of RNA is detected by 1% agarose gel electrophoresis, and the concentration and OD of RNA are detected by an ultra-micro ultraviolet spectrophotometer A260/A280 Values.
Compared with the prior art, the beneficial effects are that: the CTAB extract in the invention provides a proper buffer environment for preventing nucleic acid from being destroyed when cells are lysed. RNA is easy to be catalyzed by alkali to hydrolyze, so SSTE buffer with pH value of 7.5-8.0 is used for dissolving RNA, so that DNA and protein can enter an organic phase in the extraction process, RNA enters an aqueous phase, and RNA with higher purity can be obtained by comparison. Compared with RNA extraction kits and other RNA extraction methods, the method can fully crack the nuclease, improve the yield and purity of RNA, has high extraction efficiency, is simpler to operate compared with other hand methods, has low reagent price, and effectively reduces the economic cost.
Drawings
FIG. 1 is a formulation of an extraction buffer according to the present invention.
FIG. 2 is a schematic diagram of RNA gel electrophoresis in an embodiment of the invention.
Detailed Description
The drawings are for illustrative purposes only and are not to be construed as limiting the present patent; for the purpose of better illustrating the embodiments, certain elements of the drawings may be omitted, enlarged or reduced and do not represent the actual product dimensions; it will be appreciated by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted. The positional relationship depicted in the drawings is for illustrative purposes only and is not to be construed as limiting the present patent.
First aspect
Provides an extracting solution for extracting total RNA of a southern medicine plant, which comprises CTAB extracting buffer solution and SSTE solution,
the CTAB buffer solution comprises 20g/L PVP, 0.05mol/L tris, 0.0125mol/L EDTA, 0.055mol/L CTAB and 1mol/L NaCl;
the SSTE solution comprises 0.95mol/L NaCl, 0.02mol/L SDS, 0.01mol/L tris (pH=7.5-8.0) and 0.001mol/L EDTA;
the pH value of the tris is 7.5-8.0, and the pH value of the EDTA is 7.5-8.0.
Second aspect
Provides the application of the extract in extracting total RNA of southern medicine plants. The extracting solution is a new extracting solution formula capable of efficiently extracting RNA, and is applied to experiments for extracting total RNA of southern medicine plants, belongs to a new application, and can be widely popularized. The southernwood plant in the invention refers to: the medicinal materials of the tunnel produced in the north and south regions of Yangtze river, including all or most of the provinces of Jiangxi, fujian, hainan and the like, are commonly exemplified by patchouli, wrinkled giant hyssop, morinda root and the like.
Third aspect of the invention
The extraction method of total RNA of the southern medicine plants comprises the following steps:
s1: preparing a basic reagent and an extracting solution one day in advance, sterilizing, and preparing a gun head, a mortar and the like for sterilization;
s2: quickly grinding the south medicinal plant leaves with liquid nitrogen for no more than 5min, subpackaging into 2-tube samples after grinding, and quick-freezing the samples in liquid nitrogen;
s3: adding CTAB buffer solution and beta-mercaptoethanol into a sample, uniformly mixing on a vortex instrument, and then heating in a water bath kettle;
s4: adding chloroform and isoamyl alcohol with the volume ratio of 24:1 into each tube of sample, uniformly mixing and centrifuging, and repeating for one time;
s5: sucking the supernatant obtained in the step S4, combining the 2-tube supernatant obtained in the step S4 into 1 tube, adding LiCl, uniformly mixing, and standing for 12-15h;
s6: centrifuging the sample placed in the step S5, discarding the supernatant, adding SSTE solution, chloroform and isoamyl alcohol with the volume ratio of 24:1, uniformly mixing and centrifuging;
s7: transferring the supernatant to a new centrifuge tube, adding absolute ethanol with the same volume, standing at-80 ℃ for 30min, and centrifuging;
s8: removing supernatant, adding 1ml of 75% ethanol to wash precipitate for 1-2 times, suspending the precipitate, and centrifuging at 4deg.C for 5min at 8000 r.min;
s9: discarding supernatant, air drying, adding DEPC treated water for dissolving, and preserving for standby;
s10: RNA quality and concentration were measured.
Specifically, taking patchouli extracted from southern medicinal plants as an example, the specific experimental method is as follows:
1. early preparation
A, preparing basic reagent
1. Absolute ethyl alcohol: -20 ℃ for standing;
absolute ethanol of 2.75%;
3. DEPC water: 1L double distilled water (ultrapure water) is added into 1mL DEPC stock solution, and after 12-15h overnight, sterilization is carried out for 30min at 121 ℃;
4.0.05 mol/L tris (ph=7.5): 100mL,12.114g; (a small amount of an aqueous solution of inactivated DEPC, concentrated HCl, pH7.5, constant volume to 100 mL.)
5.0.5 mol/L EDTA, pH=7.5, 100mL;
(1) Weigh 18.91 g Na 2 EDTA·2H 2 O (mr= 374.26) was placed in a 1L beaker;
(2) About 80mL of inactivated DEPC water is added and fully stirred;
(3) NaOH adjusts pH7.5 (about 2.5g NaOH, and EDTA is completely dissolved at pH 7.5);
(4) Adding the inactivated DEPC water to a volume of 100mL, sterilizing and preserving at room temperature.
6. The CTAB extraction buffer and SSTE buffer were prepared using the above-mentioned 0.5mol/L EDTA, and reagents such as 12mol/L LiCl were prepared, as shown in FIG. 1.
The prepared solution comprises 20g/L PVP, 0.05mol/L tris, 0.0125mol/L EDTA, 0.055mol/L CTAB and 1mol/L NaCl;
SSTE solution comprises 0.95mol/L NaCl, 0.02mol/L SDS, 0.01mol/L tris (pH=7.5-8.0), 0.001mol/L EDTA;
tris had a pH of 7.5 and EDTA had a pH of 7.5.
And B, other preparation: gun head, centrifuge tube, mortar, pestle, etc., and sterilized at 121 ℃ for 30min.
2. The method comprises the following specific steps
1. Grinding: taking out herba Agastaches leaves at-80deg.C, rapidly placing into liquid nitrogen, pre-cooling the mortar and grinding rod with liquid nitrogen, sufficiently and rapidly grinding the leaves into powder, grinding for no longer than 5min, adding sample into 2 tubes (1 repeated 2 branch tubes, and finally mixing together to obtain higher concentration), and quick freezing in liquid nitrogen;
2. after all samples are ground, an ultraviolet lamp is turned on for 30min, a water bath kettle is turned on, the temperature is adjusted to 65 ℃, CTAB is placed in the water bath kettle for heating, and a centrifuge is used for precooling at 4 ℃.
3. To the centrifuge tube to which the samples were added, 0.8ml of CTAB, 50. Mu.L of beta-mercaptoethanol was added, and two tubes of extract were extracted for each sample. Mixing on vortex machine, heating in water-soluble pot at 65deg.C for 5min.
4. Chloroform and isoamyl alcohol in the volume ratio of 24:1 (0.6 mL) were added to each tube and mixed well on a vortex. (the gun head does not need to touch the centrifuge tube during sample addition) and violently oscillates for 1min, water bath is carried out for 10min, the temperature is 4 ℃, and the time is 12000 r.min -1 Centrifuging for 10min.
5. Sucking the supernatant into 2mL centrifuge tubes, adding 0.6mL chloroform and isoamyl alcohol (which can be added into new tube during centrifugation in the previous step due to tube replacement) with volume ratio of 24:1, mixing on vortex instrument, and shaking vigorously for 1min at 4deg.C and 12000 r.min -1 Centrifuging for 10min under the condition.
6. Mixing 2 tubes of supernatant into 1 tube, adding 12mol/L LiCl with 200-300 μl of supernatant volume, shaking up and down, mixing, and standing at 4deg.C overnight in refrigerator for 12-15 hr.
7. The next day, the supernatant was subjected to a temperature of 4℃and 12000 r.min -1 Centrifuging for 20 min under the condition.
8. The supernatant was discarded, the remaining supernatant was gently aspirated, and 0.4ml of SSTE buffer heated in a 65℃water bath was added, 0.4ml chloroform: isoamyl alcohol (volume ratio is 24:1), and uniformly mixing on a vortex meter, at 4 ℃ and 12000 r.min -1 Centrifuging for 10min under the condition.
9. Transferring the supernatant to a new 1.5ml centrifuge tube, adding equal volume of absolute ethanol placed at-20deg.C, mixing upside down, and placing at-70deg.C for 30min.
10. Taking out the sample, and centrifuging for 20 min under the condition of 12000 r.min at 4 ℃.
11. The supernatant was decanted, and the precipitate was washed 1-2 times with 1mL of 75% ethanol, suspended and centrifuged at 4℃for 5min at 8000 r.min.
12. The supernatant was decanted and the residual liquid was aspirated with a 200. Mu.L tip. (also can be placed on paper towel reversely and drained)
13. The hood was slightly air dried. (air-dried for 5 min)
14. Adding 20 mu L DEPC treated water for dissolving, and storing in a refrigerator at-80 ℃ for standby. (temporary storage can be carried out at-20 ℃ C. When in use)
15. Detecting RNA quality and concentration:
(1) The quality of RNA was detected by 1% agarose gel electrophoresis, 2.5. Mu.L of goldbiew reagent was added per 50mL of gum solution during gum preparation, 3. Mu.L of RNA was mixed with 1. Mu.L of 6 x Loading buffer and applied to the sample, and the gum was run under 80V,200mA electrophoresis conditions for 30min, with the electrophoresis solution being 1 XTAE solution. As shown in FIG. 1, the integrity and brightness of 28s and 18s bands were checked by photographing using a gel imager after electrophoresis was completed, the bands were bright and the edges were clear, and the quality of the extracted RNA was considered to be good.
(2) The OD values of RNA at wavelengths of 260nm and 280nm, and the concentration of RNA were determined using a Nanodrop 2000 ultra-micro uv spectrophotometer at 1 μl of RNA, and as shown in fig. 2, the comparison of the measured concentration of RNA data with conventional actual box extraction in this example, and the OD values at wavelengths of 260nm and 280nm, where OD260/OD280 = 1.7-2.2, indicate that the extracted RNA was useful.
Comparative experimental design: the general RNA extraction kit TIAN GEN RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit is purchased in the market, the total RNA is extracted from the southern medicine plants by adopting the RNA extraction kit, and the quality and the concentration of the RNA are detected by adopting the same method.
TABLE 1 test results for examples 1-8
TABLE 2 test results for comparative experiments 1-8
From the comparison of the data analysis in the table, the concentration of RNA extracted from patchouli leaves by using the extract in the example is significantly higher than that extracted by the conventional kit. Compared with the RNA extraction kit and other RNA extraction methods, the extraction solution and the corresponding extraction method provided by the invention can fully crack nuclease, improve the RNA yield and purity, have high extraction efficiency, are simpler to operate than other hand-held methods, have low reagent price, and effectively reduce economic cost. In addition, when the extraction solution and the extraction method in the embodiment are adopted to extract total RNA from other south medicinal plants, such as wrinkled giant hyssop, morinda officinalis and the like, the extraction effect and the extraction efficiency are also obviously improved compared with those of an RNA extraction kit.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. The extracting solution for extracting total RNA of southern medicine plants is characterized by comprising a CTAB buffer solution and an SSTE solution, wherein the pH values of the CTAB buffer solution and the SSTE solution are 7.5-8.0,
the CTAB buffer solution comprises 20g/L PVP, 0.05mol/L tris, 0.0125mol/L EDTA, 0.055mol/L CTAB and 1mol/L NaCl;
the SSTE solution comprises 0.95mol/L NaCl, 0.02mol/L SDS, 0.01mol/L tris and 0.001mol/L EDTA;
the pH value of the tris is 7.5-8.0, and the pH value of the EDTA is 7.5-8.0.
2. Use of the extract of claim 1 for extracting total RNA of a southern herbal plant.
3. The extraction method of the total RNA of the southern medicine plants is characterized by comprising the following steps of:
s1: preparing a basic reagent and an extracting solution one day in advance, sterilizing, and preparing a gun head and a mortar for sterilization;
s2: quickly grinding the south medicinal plant leaves with liquid nitrogen, subpackaging into 2-tube samples after grinding, and quickly freezing the samples in the liquid nitrogen;
s3: adding CTAB buffer solution and beta-mercaptoethanol into a sample, uniformly mixing on a vortex instrument, and then heating in a water bath kettle;
s4: adding chloroform and isoamyl alcohol with the volume ratio of 24:1 into each tube of sample, uniformly mixing and centrifuging, and repeating for one time;
s5: sucking the supernatant obtained in the step S4, combining the 2-tube supernatant obtained in the step S4 into 1 tube, adding LiCl, uniformly mixing, and standing for 12-15h;
s6: centrifuging the sample placed in the step S5, discarding the supernatant, adding SSTE solution, chloroform and isoamyl alcohol with the volume ratio of 24:1, uniformly mixing and centrifuging;
s7: transferring the supernatant to a new centrifuge tube, adding absolute ethanol with the same volume, standing at-80 ℃ for 30min, and centrifuging;
s8: removing supernatant, adding 1ml of 75% ethanol to wash precipitate for 1-2 times, suspending the precipitate, and centrifuging at 4deg.C for 5min at 8000 r.min;
s9: discarding supernatant, air drying, adding DEPC treated water for dissolving, and preserving for standby;
s10: RNA quality and concentration were measured.
4. The method for extracting total RNA from southern herbal plants according to claim 3, wherein in S3, CTAB extract is preheated at 65 ℃ in advance, 0.8-1ml CTAB and 50-60 μl beta-mercaptoethanol are added into a centrifuge tube to which the sample is added, and the mixture is uniformly mixed in a vortex machine and then heated in a water-soluble pot at 65 ℃ for 5min.
5. The method for extracting total RNA from southern herbal plants according to claim 4, wherein in S4, 0.6-0.8mL of chloroform and isoamyl alcohol with volume ratio of 24:1 are added into each tube, and the mixture is vigorously shaken for 1min and then water-bath is performed for 10min; the operation is repeated for 1 time, without water bath, after direct shaking, the centrifuge is precooled at 4 ℃ and centrifuged for 10min at 12000r min < -1 >.
6. The method for extracting total RNA from southern medicine plants according to claim 6, wherein in S5, 2 pipe supernatant obtained in S4 is combined into 1 pipe, 200-300 mu L of 12mol/L LiCl is added, and the mixture is mixed and placed in a refrigerator at 4 ℃ for 12-15h.
7. The method for extracting total RNA from southern herbal plants according to claim 3, wherein in S6, 0.4-0.6ml of SSTE solution is added into the tube, and the SSTE solution is heated in a water bath at 65 ℃ in advance.
8. The method for extracting total RNA from southern herbal plants according to claim 3, wherein in S7, absolute ethanol in a centrifuge tube is placed in a refrigerator at-20 ℃ for one day in advance, and after 30min at-80 ℃, at 4 ℃ and 12000 r.min -1 Centrifuging for 20 min.
9. The method for extracting total RNA from southern herbal plants according to claim 3, wherein in S9, the total RNA is dried for 5min, dissolved in 20-30. Mu.L DEPC treated water, and placed on ice to be detected or stored directly at-80 ℃.
10. The method for extracting total RNA from southern herbal plants according to claim 3, wherein in S10, the quality of RNA is detected by 1% agarose gel electrophoresis, and the concentration and OD of RNA are detected by an ultra-micro ultraviolet spectrophotometer A260/A280 Values.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492485A (en) * | 2009-03-13 | 2009-07-29 | 北京林业大学 | Method for extracting RNA in gymnosperm tissue |
| US20110300546A1 (en) * | 2010-06-08 | 2011-12-08 | Tang ke xuan | Method of utilizing the pts gene and rna interference of the ads gene to increase patchouli alcohol content in artemisia annua l. |
| CN114426965A (en) * | 2020-10-29 | 2022-05-03 | 深圳华大生命科学研究院 | Kit for simultaneously extracting DNA, RNA and protein from plant tissue sample and general method |
-
2023
- 2023-06-01 CN CN202310638959.7A patent/CN116445471A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492485A (en) * | 2009-03-13 | 2009-07-29 | 北京林业大学 | Method for extracting RNA in gymnosperm tissue |
| US20110300546A1 (en) * | 2010-06-08 | 2011-12-08 | Tang ke xuan | Method of utilizing the pts gene and rna interference of the ads gene to increase patchouli alcohol content in artemisia annua l. |
| CN114426965A (en) * | 2020-10-29 | 2022-05-03 | 深圳华大生命科学研究院 | Kit for simultaneously extracting DNA, RNA and protein from plant tissue sample and general method |
Non-Patent Citations (5)
| Title |
|---|
| 庞启华 等: "南药高良姜基因组DNA提取方法的研究", 时珍国医国药, vol. 19, no. 05, pages 1187 - 1189 * |
| 杨云 等: "濒危南药白木香愈伤组织总RNA提取方法研究", 广东农业科学, vol. 37, no. 03, pages 193 - 195 * |
| 樊青玲 等: "忽地笑不同器官RNA提取方法比较研究", 安徽农业科学, vol. 48, no. 18, pages 169 - 172 * |
| 陈英 等: "广藿香不同组织总RNA提取方法的筛选与优化", 江苏农业科学, vol. 42, no. 12, pages 54 * |
| 陶倩 等: "南方红豆杉不同组织总RNA提取方法研究", 中华中医药杂志, vol. 33, no. 08, pages 3337 * |
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