CN102181435A

CN102181435A - Method for quickly extracting ribonucleic acid (RNA) from ginseng leaf tissues

Info

Publication number: CN102181435A
Application number: CN2011101386068A
Authority: CN
Inventors: 杨成君
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2011-05-26
Filing date: 2011-05-26
Publication date: 2011-09-14

Abstract

The invention discloses a method for quickly extracting ribonucleic acid (RNA) from ginseng leaf tissues, and relates to a method for extracting the RNA. The method solves the problems that steps are complex, the consumed time is long, more medicaments are used, and the cost is high existing in the conventional method for extracting the RNA from the ginseng leaf tissues. The method comprises the following steps of: 1, grinding the ginseng leaf tissues into powder in liquid nitrogen, adding extraction buffer solution, beta-mercaptoethanol, Tris balanced phenol and chloroform, oscillating, and centrifuging; 2, taking supernatant, adding the Tris balanced phenol and the chloroform, oscillating, centrifuging, taking supernatant, adding the chloroform, oscillating, and centrifuging; 3, taking supernatant, adding isopropanol, precipitating, centrifuging, and abandoning supernatant; and 4, washing an RNA precipitate by using ethanol, and air-drying the precipitate in the air to obtain the RNA. Compared with the conventional cetyltrimethyl ammonium bromide (CTAB) method and the like, the method has the advantages that the extraction effect is higher, the time is short, and the method is easy to operate; and the extraction cost is low compared with that of a commercial kit.

Description

A kind of rapid extraction Ginseng Leaf organizes the method for RNA

Technical field

The present invention relates to the method for a kind of RNA of extraction.

Background technology

Extract the quality height from plant tissue, the RNA that integrity is good transcribes group analysis, cDNA library construction, northern engram analysis, the working foundation in the terminal rapid amplifying of DDRT-PCR, cDNA, RT-PCR equimolecular biological study field.Select suitable method most important at different vegetable materials.Because contain amounts of protein, sugar, polyphenol and other secondary metabolites meta-bolitess in the panax ginseng plant, endogenous RNase activity is higher, has seriously disturbed the extraction of RNA, causes the failure of an experiment.Most of in the past laboratories all adopt traditional CTAB method, SDS method to extract the RNA of plant, and perhaps buy the commercial reagents box and extract RNA, these method complex operation step, length consuming time, used medicine is many, the cost height.

Summary of the invention

The objective of the invention is to organize in order to solve the existing Ginseng Leaf of extraction that the method for RNA exists complex steps, length consuming time, used medicine is many and the high problem of cost, and provide a kind of rapid extraction Ginseng Leaf to organize the method for RNA.

Rapid extraction Ginseng Leaf organizes the method for RNA to carry out according to the following steps: one, the 200mg Ginseng Leaf is organized, clean to wipe away and do back grind into powder in liquid nitrogen, join then in the centrifuge tube that 750 μ L extraction damping fluid and 75 μ L beta-mercaptoethanols are housed, add 350 μ L Tris balance phenols and 350 μ L chloroforms again, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Two, get supernatant after centrifugal, add 350 μ L Tris balance phenols and 350 μ L chloroforms, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min, get supernatant after centrifugal, the chloroform that adds 750 μ L, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Three, get supernatant after centrifugal, add isopyknic Virahol, behind the mixing in-20 ℃ of precipitation 20min, centrifugal 15min under 13000r/min, 4 ℃ condition then, abandoning supernatant; Four, adopt that volumetric concentration is 75%, the ethanol 1mL washing RNA precipitation of ice bath once, dry precipitation in the air, promptly finish the rapid extraction Ginseng Leaf and organize RNA;

Wherein extract damping fluid in the step 1: 2% (quality) sodium lauryl sulphate, 4mol/L urea, 0.1mol/LNaCl, 0.01mol/L EDTA (pH8.0) and 0.05mol/L Tris-HCL (pH8.0); The volumetric concentration of beta-mercaptoethanol is 10% in the step 1.

The present invention and traditional CTAB method etc. are compared, and extraction effect is relatively good, and the time is short, easily operation.Lower than commercial reagents box extraction cost.

1, the adding of multiple composition can increase the active inhibition to RNase in the extraction damping fluid.SDS is not only protein denaturant, also is the inhibitor of RNase simultaneously, and the urea of high density also is the inhibitor of RNase.

2, before material extraction, in extracting solution, add Tris-balance phenol, chloroform in advance, more can effectively suppress the activity of RNase.Tris-balance phenol, chloroform concussion extracting simultaneously, the RNase of sex change fully removes by centrifugal.

3, added 10% beta-mercaptoethanol in the extracting solution, the beta-mercaptoethanol of high density is used for suppressing the activity of RNA enzyme, also can prevent the sample oxidation, because phenolic compound is easy to the quinine that oxidized formation connects with covalent linkage, be easier to bind nucleic acid, this has caused the irreversible destruction to RNA.Beta-mercaptoethanol can prevent the oxidation of polyphenol as strong reductant, and the disulfide linkage that can interrupt polyphenoloxidase makes it inactivation.

4, preceding 3 can solve the RNAase pollution problems.The not contaminated and degraded of protection RNA.

5, SDS is an anionic detergent, and can dissociate nucleic acid and combination of proteins, and protein combines with positively charged side chain form the SDS-protein complex and precipitate in the presence of high salt.

6, traditional CTAB method needs 65 ℃ of water-baths, does not have 65 ℃ of water-baths in the SDS method, has so also avoided in the CTAB method owing to high temperature causes the chance of polluting in RNA degraded and the water.

7, all selected high speed centrifugation (13000r/min) in all centrifugal processes, high speed centrifugation reduces centrifugation time.It is also very high to extract the RNA quality.

8, in precipitation process, in refrigerator-20 ℃, carry out the 20min precipitation, than traditional precipitation at room temperature, sedimentation effect is good, more reduces the time than the sedimentary method of spending the night, and has also reduced the also precipitated chance of other impurity on ice.Can prevent that RNA is not degraded in precipitation process.

9, about about 150 minutes, the time that the sedimentary method of spending the night needs is longer greatly for the method for tradition extraction RNA.In present method operating process, obtain time that sample needs at last within 90-100 minute from extracting.Saved the time.

10, present method is lower than commercial reagents cassette method cost, extracts test kit abroad and extracts 50 times/box, approximately wants thousands of first costs, and all ingredients used in amounts that present method preparation extraction is 50 times is wanted the cost of 500-600 unit.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of total RNA quality examination in the embodiment one; Fig. 2 is the agarose gel electrophoresis figure that RT-PCR detects in the embodiment one.

Embodiment

Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.

Embodiment one: present embodiment rapid extraction Ginseng Leaf organizes the method for RNA to carry out according to the following steps: one, the 200mg Ginseng Leaf is organized, clean to wipe away and do back grind into powder in liquid nitrogen, join then in the centrifuge tube that 750 μ L extraction damping fluid and 75 μ L beta-mercaptoethanols are housed, add 350 μ L Tris balance phenols and 350 μ L chloroforms again, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Two, get supernatant after centrifugal, add 350 μ L Tris balance phenols and 350 μ L chloroforms, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min, get supernatant after centrifugal, the chloroform that adds 750 μ L, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Three, get supernatant after centrifugal, add isopyknic Virahol, behind the mixing in-20 ℃ of precipitation 20min, centrifugal 15min under 13000r/min, 4 ℃ condition then, abandoning supernatant; Four, adopt that volumetric concentration is 75%, the ethanol 1mL washing RNA precipitation of ice bath once, dry precipitation in the air, promptly finish the rapid extraction Ginseng Leaf and organize RNA;

The Ginseng Leaf is organized as bright sample in the present embodiment step 1.

The Ginseng Leaf who extracts in the present embodiment organizes RNA, and the DEPC that adds 40 μ L can preserve standby.

The Ginseng Leaf who extracts in the present embodiment organizes RNA, carries out the RNA quality examination:

1, total RNA quality examination and analysis

1.1% agarose gel electrophoresis detects the integrity of RNA, and ultraviolet spectrophotometry is carried out purity detecting; The result as shown in Figure 1, wherein 28S, 18S band are high-visible, 28S and 18S fluorescent brightness illustrate that than near 2: 1 RNA is more complete; Get an amount of total RNA and carry out purity testing through ultraviolet spectrophotometer, the A of RNA ₂₆₀/ A ₂₈₀Value is 1.98, proves that the RNA quality is good.

2, RT-PCR detects

With the total RNA that extracts RQ1 RNase-Free DNase dna digestion; Eliminating possible DNA pollutes; Provide RT-PCR operation system specification sheets to carry out according to clontech company; Total RNA is used ThermoScript II, and reverse transcription becomes strand cDNA with SMART IV Oligonucleotide, and strand cDNA generates dscDNA by the LD-PCR amplification; The primer sequence that is provided: SMART ^TMIV Oligonucleotide (10uM): 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 '; CDSIII/3 ' PCR Primer (10uM): 5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) ₃₀N _-1N-3 ' (N=A, G, CorT; N _-1=A, G, orC); 5 ' PCRPrimer (10uM): 5 '-AAGCAGTGGTATCAACGCAGAGT-3 '; PCR program reaction parameter is as follows: 95 ℃ of pre-sex change 1min; 95 ℃ of sex change 15s, 68 ℃ are extended 6min, totally 23 circulations; Finish to react to 4 ℃.Getting 5 μ L reaction product carries out 1.1% agarose electrophoresis and identifies product.

The result as shown in Figure 2, through the detection of 1.1% agarose gel electrophoresis, the RNA that the SDS method is extracted obtains the disperse of ds cDNA fragment band through RT-PCR and distributes size at 0.2-3.0kb, shows that further total RNA of present embodiment extraction is up-to-standard, degradation amount is few, and the long segment of cDNA is more; Illustrate can be used in and carry out next step cDNA library construction, transcribe the biological study of group order-checking equimolecular.

Embodiment two: present embodiment and embodiment one are different is that the specification of centrifuge tube in the step 1 is 1.5ml or 2.0ml.Other step and parameter are identical with embodiment one.

Embodiment three: what present embodiment and embodiment one were different is that Virahol will carry out precooling before use in the step 3, and precooling temperature is-20 ℃.Other step and parameter are identical with embodiment one.

Claims

1. a rapid extraction Ginseng Leaf organizes the method for RNA, it is characterized in that the rapid extraction Ginseng Leaf organizes the method for RNA to carry out according to the following steps: one, the 200mg Ginseng Leaf is organized, clean to wipe away and do back grind into powder in liquid nitrogen, join then in the centrifuge tube that 750 μ L extraction damping fluid and 75 μ L beta-mercaptoethanols are housed, add 350 μ L Tris balance phenols and 350 μ L chloroforms again, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Two, get supernatant after centrifugal, add 350 μ L Tris balance phenols and 350 μ L chloroforms, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min, get supernatant after centrifugal, the chloroform that adds 750 μ L, behind the concussion 5min under 13000r/min, 4 ℃ condition centrifugal 5min; Three, get supernatant after centrifugal, add isopyknic Virahol, behind the mixing in-20 ℃ of precipitation 20min, centrifugal 15min under 13000r/min, 4 ℃ condition then, abandoning supernatant; Four, adopt that volumetric concentration is 75%, the ethanol 1mL washing RNA precipitation of ice bath once, dry precipitation in the air, promptly finish the rapid extraction Ginseng Leaf and organize RNA;

Wherein extract damping fluid in the step 1: 2% (quality) sodium lauryl sulphate, 4mol/L urea, 0.1mol/L NaCl, 0.01mol/L EDTA and 0.05mol/L Tris-HCL; The volumetric concentration of beta-mercaptoethanol is 10% in the step 1.

2. a kind of rapid extraction Ginseng Leaf according to claim 1 organizes the method for RNA, and the specification that it is characterized in that centrifuge tube in the step 1 is 1.5ml or 2.0ml.

3. a kind of rapid extraction Ginseng Leaf according to claim 1 organizes the method for RNA, it is characterized in that Virahol will carry out precooling before use in the step 3, and precooling temperature is-20 ℃.