The extraction and purification of intact RNA is a prerequisite for RNA research, such as Nothern hybridization, mRNA isolation, RT-PCR, quantitative PCR, cDNA synthesis and in vitro translation. There are five key points in the extraction process of all RNAs, namely: 1) efficient fragmentation of sample cells or tissues; 2) effective denaturation of nucleoprotein complexes; 3) effective inhibition of endogenous RNases; Effectively separating RNA from DNA and protein mixtures; 5) Effective removal of polysaccharide impurities is also involved for samples with high polysaccharide content. But the most critical of these is the inhibition of RNase activity. At the present stage of RNA extraction, two routes can be used. 1) Extracting total nucleic acids and precipitating RNA with lithium chloride; 2) Extracting directly under acidic conditions, DNA and protein entering the organic phase under acidic conditions and RNA Stay in the water phase. The first method of extraction will result in the loss of small molecular weight RNA, which is currently used at a low frequency.
Method 1: Rapid extraction of total RNA kits
Some companies have introduced total RNA extraction kits that can be used to prepare high quality RNA for building libraries. The total RNA purification system uses two well-known RNase inhibitors, isothiocyanate arc (GTC) and β-mercaptoethanol, plus the entire operation is carried out in an ice bath, which significantly reduces the rate of RNA degradation. The combination of GTC and sodium N-dodecyl sarcosinate will facilitate the dissociation of the nucleoprotein complex, separate the RNA from the protein, and release the RNA into the solution. Further purification of RNA from the complex was carried out according to a one-step rapid extraction method of Chomc zynski and Sacchi, using an acidic phenol-chloroform mixture. Low pH phenols will cause RNA to enter the aqueous phase, thus separating it from proteins and DNA that remain in the organic phase. The RNA in the aqueous phase can be concentrated by isopropanol precipitation. Further, the above RNA precipitate is re-dissolved in the GTC solution, followed by secondary precipitation with isopropanol, followed by washing the precipitate with ethanol to remove all residual proteins and inorganic salts, and if the inorganic salt is contained in the RNA, it is possible Inhibition of some enzymatic reactions in later operations.
One: instrument
Constant temperature water bath, frozen high speed centrifuge, ultraviolet spectrophotometer, liquid taker, electrophoresis instrument, electrophoresis tank.
Two: reagent
RNA extraction kit, 0.05% diethylpyrocarbonate (DEPC), 75% ethanol
Steps
(a): broken cells or tissues
A: Microbial materials
1: Fermentation of bacteria in 3-4 days (or logarithmic growth phase), collecting mycelium by centrifugation (plant and animal materials do not need to be treated in this step)
2: Wash the mycelium 2-3 times with DEPC-treated water, and remove the remaining water as much as possible.
3: Add liquid nitrogen to grind it to make it a powder to release RNA
4: The ground sample was transferred to a container of 12 ml of denaturant and homogenized.
B: Animal and plant cell culture material (applicable sample amount is cell: 108)
1: cell or tissue culture: according to conventional methods
2: Breaking of deep suspension culture cells:
(1) Cell collection: The cell culture medium containing a certain concentration is placed in a sterile centrifuge tube, centrifuged at 3000 ° for 5 minutes at 4 ° C.
(2) Cell washing: The supernatant was washed with 25 ml of sterilized frozen 1×PBS buffer, and then centrifuged at 3000 g for 5 minutes at 4 ° C.
(3) Cell disruption: 15 ml of pre-cooled denaturing solution was added to the pelleted cells and homogenized with a sterile treated homogenizer.
3: Breakage of surface culture cells:
(1) Determine the number of culture flasks, so that the total number of cells selected for each flask is increased by 108.
(2) Cell collection: The culture solution was poured out, washed once with pre-cooled sterile PBS buffer, 8 ml of pre-cooled denaturing solution was added to the culture flask, and the culture flask was rotated to dissolve the cells, and the viscosity was increased. .
(3) Using a sterile pipette, draw the liquid from the first flask into the second flask, spin, dissolve the cells, inhale the third flask, and so on, until all the selected flasks are washed. Once, then add 4 ml of the above denaturant from the first flask and wash all the flasks once.
(4) The above 12 ml of the cell-containing denaturing solution was transferred to a 50 ml sterile centrifuge tube, and the cells were homogenized and disrupted.
C: Plant tissue disruption (applicable sample size is 0.05g tissue)
(1) 600 ul of the denatured liquid was placed in a 1.5 ml centrifuge tube, and the tube was placed in an ice bath for 5 minutes.
(2) Freezing 0.05g of fresh tissue with liquid nitrogen
(3) grinding the tissue block under liquid nitrogen
(4) After the liquid nitrogen is volatilized, the above dispersed tissue is transferred to a sterile centrifuge tube.
D: Animal tissue is broken (applicable sample size is 1 gram of tissue)
(1) 12 ml of the denaturing solution was placed in a 50 ml centrifuge tube, and the tube was placed in an ice bath for 5 minutes.
(2) Add 1 gram of fresh or frozen animal tissue to the above tube and homogenize and pulverize.
Note 1: The sample used for RNA extraction of the tissue block must be fresh cells or tissue. If it is not immediately available for extraction after sampling, the sample should be stored in a refrigerator at -70 °C.
2: The denaturing solution and the corresponding centrifuge tube need to be pre-cooled.
(2): Extraction of RNA
The cells or tissues homogenized by the denaturant are thoroughly mixed with 600 ul + 60 ulp H4.0 of 2 M sodium acetate, and a vortex shaker can be used.
600 ul phenol \ chloroform \ isoamyl alcohol (25 \ 24 \ 1), thoroughly mixed or vortex shaker for 10 seconds.
Bingyu 10-15 minutes, centrifugation, 4 ° C, 10000 g, 20 minutes.
The upper aqueous phase was pipetted into a sterile centrifuge tube + an equal volume of isopropanol, and the RNA was precipitated at -70 ° C for 30 minutes.
Centrifuge at 4 ° C, 10000 g, 20 minutes.
The RNA was precipitated, lyophilized for 15 minutes, and the RNA pellet was redissolved in 300 ul of the denaturant and oscillated (sometimes to help dissolve, it can be heated at 65 ° C, but the time should be extremely short).
Mix 60 ulH4.0 2M sodium acetate thoroughly and use a vortex shaker.
600 ul phenol \ chloroform \ isoamyl alcohol (25 \ 24 \ 1), thoroughly mixed or vortex shaker for 10 seconds.
Bingyu 10-15 minutes, centrifugation, 4 ° C, 10000 g, 20 minutes.
The upper aqueous phase is aspirated into a sterile centrifuge tube.
Equal volume of isopropanol secondary precipitation (-70 ° C, 30 minutes), 75% ethanol wash precipitation, precipitation freeze-dried, redissolved in deionized water (for the preparation of long-term preservation of RNA can be added to pH 5.0 sodium acetate to The final concentration was 0.25 M, and then 2.5 volumes of ethanol were added and stored at -70 ° C.)
Note: 1 composition of denaturant: 25g guanidinium isothiocyanate dissolved in 33ml CSB (42mM sodium acetate, 0.83% sodium lauryl sarcosinate, 0.2mM β-mercaptoethanol), dissolved at 65 ° C, filter sterilization , pre-cooled at 4 ° C.
2 Acid phenol preparation: At 55 ° C, 500 g of phenol was dissolved in 500 ml of 50 mM, pH 4.0 sodium acetate, mixed, and after static stratification, the supernatant was removed, and 500 ml of 50 mM, pH 4.0 sodium acetate was added repeatedly until the pH was <4.1.
Method 2: Extraction of total RNA by lithium chloride
The method uses high concentration of urea denatured protein and inhibits RNase, and selects precipitated RNA with lithium chloride. It is especially suitable for extracting a small amount of tissue RNA from a large number of samples, which has the advantages of quick and simple, but also has a small amount of DNA contamination and RNA. The rate is not high and the defects of small RNA fragments are lost.
Instrument: Same as above
Reagents:
Lithium chloride/urea solution [lithium chloride 126g (3M) urea, 360g (6M), add water to 1L, filter sterilization]
Suspension [10 mM Tris-HCL (pH 7.6), 1 mM EDTA (pH 8, 0), 0.5% SDS]
The rest is the same as above
Steps:
1: For a large number of tissues or cells, add 5-10 ml of lithium chloride-urea solution per gram of tissue or cells, and homogenize the paddle for 2 minutes at high speed; for a small amount of cells (107 cells/ml), per gram of tissue or The cells were manually padded by adding 0.5 ml of lithium chloride-urea solution and transferred to an Eppendof tube.
2: The slurry is placed at 0-4 ° C for 4 hours, and centrifuged at 12000 g for 30 minutes.
3: Take the precipitate, add 1/2 volume of lithium chloride-urea solution to the original slurry, repeat step 2
4: Precipitation is reconstituted with 1/2 volume of lithium chloride-urea solution. Add an equal volume of phenol/chloroform/isoamyl alcohol at room temperature for 15-30 minutes, then shake and mix, and centrifuge at 4000 g for 5 minutes.
5: The upper aqueous phase was taken, and 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol were added, and the mixture was allowed to stand at -20 ° C for 1 hour, and centrifuged at 5000 g.
6: 70% ethanol wash precipitate once. Dry in vacuum.
7: The RNA lysate was dissolved and precipitated, and RNA was obtained and stored at -70 ° C.
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