CN102409040A

CN102409040A - Method for extracting dendrobium total RNA (Ribonucleic Acid)

Info

Publication number: CN102409040A
Application number: CN2011103702680A
Authority: CN
Inventors: 徐祥彬; 王慧中; 宋红苗; 史学群
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2011-11-18
Filing date: 2011-11-18
Publication date: 2012-04-11

Abstract

The invention discloses a method for extracting dendrobium total RNA (Ribonucleic Acid). The method comprises the following steps of: precipitating polysaccharide by using potassium acetate (pH of 4.8, 5M); favorably precipitating the polysaccharide to a lower layer by high-concentration K<+> under the condition of lower pH and remaining RNA in upper layer solution to remove a part of polysaccharide in the RNA and cause high-concentration K<+> in upper layer RNA liquid phase so as to facilitate subsequent extraction of chloroform; further removing polysaccharide ingredient in the RNA, wherein the great majority of polysaccharide ingredient in the RNA can be removed after the two steps; and finally, performing LiCl precipitation to obtain a large amount of RNA with higher quality. The method is easy to operate, low in extraction cost and good in repeatability, and high-quality and high-concentration RNA can be obtained.

Description

A kind of method of extracting the total RNA of the stem of noble dendrobium

(1) technical field

The present invention relates to the method that the total RNA of a kind of plant tissue extracts, particularly a kind of method of extracting the total RNA of the stem of noble dendrobium.

(2) background technology

Dna purity RNA high, good in integrity carries out the research of molecular biology aspect from plant tissue, as cDNA synthesize, the prerequisite and the key of Northern hybridization, mRNA separation, RT-PCR, cDNA library construction and external translation etc.Many biomaterials all can run into the pollution problem of polysaccharide when extracting nucleic acid.Many physico-chemical properties of polysaccharide are very similar with RNA, are difficult to they are separated, and RNA is also wrapped up in easily and carried off when removing polysaccharide, causes the minimizing of RNA output.When precipitated rna, also be easy to generate the RNA gelatinous precipitate that contains polysaccharide, this RNA deposition is insoluble in water, or the dissolving back produces thick solution.In addition, polysaccharide can also suppress the activity of many enzymes, and the RNA sample that has therefore polluted polysaccharide can't be used for further molecular biology research.

In the RNA of routine process for extracting, the SDS-Guanidinium hydrochloride is handled can remove a part of polysaccharide; Under high density Na+ or the K+ ion existence condition, can remove some polysaccharide through phenol, chloroform extracting; Low-concentration ethanol can precipitate the part polysaccharide; Also can the part polysaccharide be stayed in the supernatant through the LiCl precipitated rna.Even but can find still that through these steps considerable polysaccharide is mingled in RNA, so polysaccharide pollution problems also need solve the plant RNA separation and purification time with more efficient methods.

(3) summary of the invention

The object of the invention provides a kind of method of extracting the total RNA of the stem of noble dendrobium, and this method is simple to operate, and extraction cost is low, and good reproducibility can obtain the RNA of high quality, high density.

The technical scheme that the present invention adopts is:

A kind of method of extracting the total RNA of the stem of noble dendrobium, described method is:

(1) the fresh stem of noble dendrobium stem that will gather places the liquid nitrogen grind into powder, changes in the centrifuge tube that diethylpyrocarbonate (DEPC) was handled, and adds to contain in the RNA extracting solution of water saturation phenol; Thermal agitation 5～10min behind the mixing; Add chloroform again, vibration, extracting 5～10min; The centrifugal 15min of 5000rpm under the room temperature gets supernatant A; The said volumetric usage that contains water saturation phenol is 30% of a RNA extracting liquid volume consumption; Said RNA extracting solution is made up of following component: the 50mMTris-Cl of pH9.0,100mM NaCl, 1%SDS;

(2) getting supernatant A that step (1) obtains, to add pH be 4.8 KAc solution, and the concentration of KAc solution is 5mol/L, and fully mixing is placed 15～30min for 4 ℃, 4 ℃ of centrifugal 20min of following 12000g, supernatant B; Said KAc liquor capacity consumption is 1/3 of a supernatant A volume;

(3) get the supernatant B that step (2) obtains, add the equal-volume chloroform, use forced oscillation, extracting 5～10min, the centrifugal 15min of 5000rpm gets supernatant under the room temperature, repeats extracting 3～5 times, the final supernatant C that gets;

(4) get the supernatant C that step (3) obtains, add the LiCl of 1/3 volume 8M, mixing, in 4 ℃ of placement 16h～20h precipitated rnas, 4 ℃ of centrifugal 20min of following 10000rpm get deposit D then;

(5) get the deposit D that step (4) obtains, with 70% washing with alcohol after, dry up the deposition after the washing, handle the deposition after double distilled water (ddH2O) dissolving dries up with DEPC, obtain the total RNA of the said stem of noble dendrobium, preservation is subsequent use in-80 ℃.

Further, the said equal-volume chloroform of preferred steps (3) repeats extracting 3 times.

The stem of noble dendrobium according to the invention is dendrobium (Dendrobium), picks up from medicinal plant germplasm improvement of Hangzhou Pedagogic University Zhejiang Province and key points of quality control laboratory.

The present invention is through the extracting solution isolation of RNA, and (pH4.8 5M) precipitates polysaccharide to Potassium ethanoate; Remove albumen and part polysaccharide through 3 times chloroform repetition extracting again, pass through LiCl (8M) precipitated rna at last, the RNA good in integrity of gained; Purity is high, and the quantity of the RNA of extraction is also bigger.The RNA sample that extracts can satisfy basic molecular biology experiment requirement, can be used for carrying out RT-PCR and Northern hybridization.

Compared with prior art, beneficial effect of the present invention is mainly reflected in: the present invention utilize earlier Potassium ethanoate (pH4.8,5M) deposition polysaccharide, under lower pH, the K of high density ⁺Help polysaccharide precipitation to lower floor, RNA then is retained in upper solution, thereby removes the part polysaccharide among the RNA, and causes the K of upper strata RNA liquid phase middle and high concentration ⁺, help chloroform extracting subsequently, further remove the polysaccharide component among the RNA, through after above two steps, the polysaccharide component among the RNA can be removed greatly, the last RNA that can obtain better quality and quantity again through the LiCl deposition; The present invention is simple to operate, and extraction cost is low, and good reproducibility can obtain the RNA of high quality, high density.

(4) description of drawings

Fig. 1 agarose gel electrophoresis detects stem of noble dendrobium total tissue RNA, behind the electrophoresis with the take pictures design sketch of gained of gel imaging system: A is a well, and B is 28S RNA, and C is 18S RNA, and D is 5S RNA.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1

1, the preparation of experimental drug:

RNA extracting solution preparation (1000ml): 1M HCl:7ml; Tris alkali: 6.05g; NaCl:5.85g; SDS:10g adds water and is settled to 1000ml.

8M LiCl is (with the 0.1%DEPC-H of sterilization ₂The O preparation)

5M KAc (pH4.8) is (with the 0.1%DEPC-H of sterilization ₂The O preparation)

2, experimental article is handled:

Glass in the leaching process and metalware toast 8h continuously at 180 ℃; Plastic centrifuge tube and rifle head are all used 0.1%DEPC-H ₂O soaks, then autoclave sterilization; Electrophoresis chamber and comb are used hydrogen peroxide dipping 30min, use aseptic water washing again, use 0.1%DEPC-H at last ₂The O flushing.

3, experimental implementation:

The fresh stem of noble dendrobium stem 5g that (1) will gather places the liquid nitrogen grind into powder; Change in the centrifuge tube that diethylpyrocarbonate (DEPC) was handled, add and contain among the above-mentioned RNA extracting solution 10ml of water saturation phenol thermal agitation 5min behind the mixing; Add chloroform again; Vibration, extracting 5min, the centrifugal 15min of 5000rpm under the room temperature; The said volumetric usage that contains water saturation phenol counts 30% with the RNA extracting liquid volume;

(2) get supernatant adding KAc (5mol/L, pH=4.8) abundant mixing under the condition, 4 ℃ of placement 15～30min, 4 ℃ of centrifugal 20min of following 12000g that step (1) obtains; Said 5mol/L KAc volumetric usage is 1/3 of a supernatant volume;

(3) get the supernatant that step (2) obtains, add the equal-volume chloroform, use forced oscillation, extracting 5min, the centrifugal 15min of 5000rpm gets supernatant under the room temperature, repeats extracting 3 times;

(4) get the supernatant that step (3) obtains, add the LiCl of 1/3 volume 8M, mixing is placed 16h～20h precipitated rna, 4 ℃ of centrifugal 20min of following 10000rpm then in 4 ℃;

(5) get the deposition that step (4) obtains, after 70% washing with alcohol, dry up deposition, handle double distilled water (ddH2O) dissolution precipitation, obtain the total RNA of the said stem of noble dendrobium, in-80 ℃, preserve subsequent use with DEPC.

4, RNA integrity detection

Get 2 μ l RNA of method for preparing, 1% agarose gel electrophoresis detects its integrity.Can observe more complete RNA through gel imaging, wherein the brightness of 28S RNA approximately is the twice of 18S RNA, and well is normal, does not have shinny conditions of streaking (Fig. 1).

5.RNA quality examination

Get 2 μ l RNA of method for preparing, be diluted to 100 μ l with DEPC water after, (USA) ratio of measure R NA concentration and A260/A280 in the sample table is analyzed the RNA quality for Thermo Scientific, Wilmington to join Nanadrop.The result shows that the RNA that in the higher stem of noble dendrobium stem of sugar degree, extracts with present method has higher quality and output, A260/A280=1.96 ± 0.07 wherein, and output is 30 ± 2.9 μ g/g fresh weights.

Claims

1. method of extracting the total RNA of the stem of noble dendrobium is characterized in that described method is:

(1) the fresh stem of noble dendrobium stem that will gather places the liquid nitrogen grind into powder, changes in the centrifuge tube that diethylpyrocarbonate was handled, and adds to contain in the RNA extracting solution of water saturation phenol; Thermal agitation 5～10min behind the mixing; Add chloroform again, vibration, extracting 5～10min; The centrifugal 15min of 5000rpm under the room temperature gets supernatant A; Said RNA extracting solution is made up of following component: the 50mM Tris-Cl of pH9.0,100mM NaCl, 1%SDS;

(5) get the deposit D that step (4) obtains, with 70% washing with alcohol after, dry up the deposition after the washing, handle the deposition after the double distilled water dissolving dries up with diethylpyrocarbonate, obtain the total RNA of the said stem of noble dendrobium, preservation is subsequent use in-80 ℃.

2. the method for the total RNA of the extraction stem of noble dendrobium as claimed in claim 1, other are that the said volumetric usage that contains water saturation phenol of step (1) is 30% of a RNA extracting liquid volume.

3. the method for the total RNA of the extraction stem of noble dendrobium as claimed in claim 1, other are that the said equal-volume chloroform of step (3) repeats extracting 3 times.