CN101974512B

CN101974512B - Tomato total RNA extraction method suitable for microRNAs analysis

Info

Publication number: CN101974512B
Application number: CN2010105210154A
Authority: CN
Inventors: 杨迎伍; 李正国; 吴玉; 邓伟
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2010-10-27
Filing date: 2010-10-27
Publication date: 2012-08-22
Anticipated expiration: 2030-10-27
Also published as: CN101974512A

Abstract

The invention provides a tomato total RNA extraction method suitable for microRNAs analysis, which is characterized in that by the step of extracting total RNA through 80-DEG C preheated phenol and a TLES buffer solution (100mM tris(hydroxymethyl)aminomethane (Tris pH 8.0), 10mM lithium chloride, 10mM ethylene diamine tetraacetic acid (EDTA pH 8.0), 1 percent sodium dodecyl sulfonate (SDS, w/v)) by combining isopropyl alcohol and sodium acetate/alcohol, large-segment RNA can be separated, and micromolecular RNA can be effectively settled. The extracted RNA has high quality and good integrity, and can be used for the gene separation and expression analysis of RT-PCR, northern hybrid and the like, and the research of micromolecular RNA of microRNAs and the like. The method has the advantages of simpleness, low cost, high efficiency and the like and is easy to operate and suitable for being used in a molecular biology experiment.

Description

A kind of tomato method for extracting total RNA that is applicable to that microRNAs analyzes

One, technical field

The present invention relates to a kind of plant method for extracting total RNA, particularly relate to the method for from the tomato tissue, extracting total RNA.

Two, background technology

It is to cause RNA to extract difficult one of the main reasons that the RNA enzyme pollutes, and the RNA enzyme has two kinds of sources usually, i.e. exogenous rna enzyme and endogenous RNA enzyme.Vessel, reagent and solution, and researchist etc. itself that the former prepares process from RNA, can through such as DEPC water reagent preparation with handle vessel, and the cleaning of maintenance environment and operator's duty are changed the influence that mode such as gloves reduces the exogenous rna enzyme.Carry and endogenous RNA enzyme is a vegetable material itself, have only through improving process for extracting itself to overcome.Because the ubiquity and the high stability thereof of RNA enzyme bring bigger difficulty often for the separation of high quality RNA.So setting up good RNA extractive technique is the prerequisite of molecular biology of plants research.Tomato is one of most important vegetable variety in the whole world, also is simultaneously the important model plant of molecular biology of plants research, especially in fruit development and ripe regulation analyse, has irreplaceable status.Through the development of several years, though formed the extractive technique of the total RNA of some tomatoes, like guanidine isothiocyanate method, these traditional methods generally consumption to reagent and experiment material are big, the cost height, and operate careless slightlyly, will cause the degraded of RNA.Especially less experiment usually causes experiment material and the unnecessary waste of reagent for the RNA demand.Have good extraction effect though RNA extracts test kit, like the RNeasy Plant Mini Kit of QIAGENE company, the price that test kit is expensive is often brought bigger economic pressures to research work, is difficult to generally applied.

MicroRNAs (miRNAs) is the non-coding endogenous RNA between 21-25nt (nucleotides), and Eukaryotic genetic expression is had very important regulating and controlling effect, is present molecular biological forward position and hot fields.In miRNA research, high-quality RNA is the prerequisite that miRNA hybridization, quantitative PCR etc. are analyzed.Because the fragment of miRNA is too short; In RNA leaching process, lose serious based on centrifugal post adsorption and purification method; So TRIZOL (Invitrogen) and TRI reagent (Sigma) etc. are widely used in the RNA that little RNA such as miRNA analyzes to be separated based on the extractive technique of coprecipitation mode.

The object of the present invention is to provide a kind of simple, economic, tomato method for extracting total RNA in a small amount efficiently, for the researchist provides more selection in real work.The RNA quality of being extracted is high, can satisfy gene isolation and expression analysis such as RT-PCR, Northern hybridization, and the needs of microRNA research, can reduce the cost of experiment simultaneously greatly.

Three, summary of the invention

For realizing above-mentioned purpose, the operation steps that the present invention adopted is:

1) tomato organization material fresh or freezing preservation fully is ground into powder in liquid nitrogen.

2) in centrifuge tube, add isopyknic phenol and TLES damping fluid; This damping fluid contains 100mM Tutofusin tris (Tris; PH 8.0), 10mM lithium chloride, 10mM YD 30 (EDTA, pH 8.0) and volume ratio be 1% sodium lauryl sulphate (SDS), in 80 ℃ of abundant preheatings.

The tomato plants material of 3) in liquid nitrogen, pulverizing in right amount changes over to aforementioned tube, and behind 80 ℃ of insulation 30s, violent vortex 1min.

4) add the chloroform/primary isoamyl alcohol of 1/2 volume then, the volume ratio of two kinds of compositions is 24: 1 in this mixed solution, fully behind the vortex mixing, and 4 ℃, 12, the centrifugal 5min of 000g is transferred to supernatant in the one new centrifuge tube.

5) use isopyknic chloroform/primary isoamyl alcohol (volume ratio is 24: 1) extracting more once, supernatant goes to a new centrifuge tube, adds isopyknic Virahol, behind the precipitation at room temperature 30min, and 4 ℃, 12, the centrifugal 10min of 000g removes supernatant.

6) resolution of precipitate adds the 3mol/L sodium acetate of 1/10 volume and the absolute ethyl alcohol of 2 times of volumes again in an amount of DEPC water, after-80 ℃ of depositions 1h or-20 ℃ spend the night, and 4 ℃, 12, the centrifugal 30min of 000g removes supernatant.

7) the RNA deposition is after 70% ethanol is washed twice, and drying is dissolved in an amount of DEPC water, and-20 ℃ of preservations are subsequent use.

The invention advantage of " the tomato method for extracting total RNA that a kind of microRNAs of being applicable to analyzes " is, can carry out a small amount of to the RNA of tomato tissue and extract, and practices thrift experimental cost greatly.For example, phenol and TLES damping fluid respectively the usage quantity of 500 μ L can effectively extract total RNA (table 1, accompanying drawing 1) of 0.04-0.10g tomato leaf, OD _260/280Be about 1.8, explain that the RNA purity of extracting is higher.Though the yield that this inventive method is extracted can satisfy the demand of molecular biology research fully a little less than TRIZOL (Invitrogen company).This inventive method can also effectively precipitate small fragment RNA simultaneously, and the RNA that is extracted not only can be used for gene isolation and expression analysis, and can be applicable to the research (accompanying drawing 2) of microRNAs such as microRNAs.

Purity and the yield of the total RNA of table 1

Annotate: MV ± deviation is from 3 repeated experiments.

Four, description of drawings

Fig. 1. total RNA gel electrophoresis figure that present method and TRIZOL (Invitrogen company) extract.

Swimming lane

1,2 and 3 is respectively total RNA that 0.04g, 0.07g and 0.10g vanes present method are extracted, total RNA that swimming lane 4 extracts for the Trizol method.

Fig. 2. present method and TRIZOL (Invitrogen company) extract the microRNA northern hybridization figure of RNA.Ladder is the Marker of microRNA, and 1 and 2 are respectively the sly-miR167 hybridization band of present method and Trizol method extraction RNA.EtBr stain is the RNA electrophorogram, equates to show 1 and 2 RNA amount.

Five, embodiment

Embodiment one: in the 5mL centrifuge tube, add 500 μ L phenol and 500 μ L TLES damping fluids, in 80 ℃ of abundant preheatings.0.07g the tomato leaf material of in liquid nitrogen, pulverizing changes over to aforementioned tube, and in 80 ℃ of insulation 30s, violent vortex 1min.Chloroform/the primary isoamyl alcohol that adds 1/2 volume again, behind the abundant vortex mixing, 4 ℃, 12, the centrifugal 5min of 000g.Supernatant is transferred in the new centrifuge tube, uses again that isopyknic chloroform/the primary isoamyl alcohol extracting once.Supernatant goes to a new centrifuge tube, adds isopyknic Virahol, behind the precipitation at room temperature 30min, and 4 ℃, 12, the centrifugal 10min of 000g.Remove supernatant, resolution of precipitate adds the 3mol/L sodium acetate of 1/10 volume and the absolute ethyl alcohol of 2 times of volumes in an amount of DEPC water.After-80 ℃ of depositions 1h or-20 ℃ spend the night, 4 ℃, 12, the centrifugal 30min of 000g removes supernatant.The RNA deposition is after 70% ethanol is washed twice, and drying is dissolved in an amount of DEPC water, and-20 ℃ of preservations are subsequent use.

Embodiment two: the usage quantity through suitable increase reagent is extracted more a large amount of total RNA of vegetable material.Adding 5mL phenol and 5mL TLES damping fluid are used to extract 0.5g tomato leaf material in the 50mL centrifuge tube, and operation steps is with embodiment one.

Total RNA effect that above embodiment extracted is seen table 1 and Figure of

description

1,2.

Claims

1. tomato method for extracting total RNA that is applicable to that microRNAs analyzes is characterized in that following operation:

A, in centrifuge tube, add isopyknic phenol and TLES damping fluid, and in 80 ℃ of abundant preheatings;

B, the tomato plants material of in liquid nitrogen, pulverizing in right amount change over to aforementioned tube, and behind 80 ℃ of insulation 30s, violent vortex 1min; Add and the isopyknic chloroform/primary isoamyl alcohol of above-mentioned phenol usage quantity then; Behind the abundant vortex mixing, 4 ℃, 12; The centrifugal 5min of 000g is transferred to supernatant in the one new centrifuge tube;

C, use with the isopyknic chloroform of supernatant/primary isoamyl alcohol extracting once, supernatant is gone to a new centrifuge tube, add again and the isopyknic Virahol of supernatant, behind the precipitation at room temperature 30min, 4 ℃, 12, the centrifugal 10min of 000g removes supernatant;

D, resolution of precipitate add the 3mol/L sodium acetate of 1/10 times of volume and the absolute ethyl alcohol of 2 times of volumes again in an amount of DEPC water, after-80 ℃ of depositions 1h or-20 ℃ spend the night; 4 ℃, 12, the centrifugal 30min of 000g; Remove supernatant; The RNA deposition is after 70% ethanol is washed twice, and drying is dissolved in an amount of DEPC water;

TLES damping fluid component is: 100mM Tutofusin tris, 10mM lithium chloride, 10mM YD 30,1% sodium lauryl sulphate; The pH value is 8.0; Wherein, Quality and the ratio of TLES damping fluid volume of 1% expression sodium lauryl sulphate, the quality of sodium lauryl sulphate in g, TLES damping fluid volume in mL; Chloroform in chloroform/primary isoamyl alcohol solution and primary isoamyl alcohol volume ratio are 24: 1; In above-mentioned steps b, add chloroform/primary isoamyl alcohol after, the volume ratio of contained TLES damping fluid, phenol, chloroform, primary isoamyl alcohol is 25: 25: 24 in the solution: 1.

2. method for extracting total RNA as claimed in claim 1 is characterized in that used experiment material is tomato leaf, flower, fruit, a root fresh, that the children is tender.

3. method for extracting total RNA as claimed in claim 1 is characterized in that employed TLES damping fluid and sodium acetate solution are the preparation of DEPC water.

4. method for extracting total RNA as claimed in claim 1 is characterized in that the usage ratio of phenol, TLES damping fluid and chloroform/primary isoamyl alcohol among the step b is 1: 1: 1, and can suitably increase the consumption of phenol, TLES and chloroform/primary isoamyl alcohol along with the increase of experiment material; Chloroform in chloroform/primary isoamyl alcohol solution and primary isoamyl alcohol volume ratio are 24: 1.

5. method for extracting total RNA as claimed in claim 1; The a small amount of that it is characterized in that both can carrying out RNA is extracted the extracted amount that also can enlarge RNA through the usage quantity of amplifying reagent; Through the consumption of appropriate change extracting solution, the tomato material of 0.04～0.5g all is its effective extraction scopes.

6. method for extracting total RNA as claimed in claim 1 is characterized in that this method can effectively precipitate small fragment RNA, and total RNA of extraction can be used for the research of microRNAs microRNA.