Liver cell RNA extraction method
one. principle
RNA extraction technology is not only an important part of molecular biology technology, but also an important basis for functional genomics research and technology. Studying the regulation mechanism of genes in organisms from the RNA level has become an important means of molecular biology research. To study the traits of a certain organism or tissue, the trait gene is first obtained. The isolation of intact RNA from tissue cells is crucial for experiments such as molecular cloning and gene expression analysis, such as Northern blotting and hybridization analysis, cDNA synthesis, etc. The effectiveness of the experiment depends to a large extent on the quality of the RNA. Using extracted RNA, one can quantitatively detect specific gene expression and accurately understand the laws of cell life activities at the molecular level. Usually a typical mammalian cell contains about 10-5 μg of RNA, of which 80%-85% are rRNA (mainly 28S, 18 S and 5.8 S, 5S four types); 10% to 15% are tRNA and intranuclear small molecules RNA.
The size and sequence of these peak RNAs can be determined by gel electrophoresis, density gradient centrifugation, anion exchange chromatography, and high pressure liquid chromatography (HPLC). In contrast, 1% to 5% of the total RNA is mRNA. Although mRNAs vary in size and nucleotide sequence, ranging from hundreds to thousands of bases, most eukaryotic mRNAs have a tail of oligoadenylate (polyA) at their 3' end. The length is generally sufficient to adsorb to oligo(dT), which allows the mRNA to be separated by affinity chromatography. This population encodes all of the polypeptides synthesized by this cell. Studies have shown that RNA is extremely unstable and easy to degrade, while RNase is almost ubiquitous and particularly stable. Therefore, the key factor in extracting RNA is to avoid the contamination of exogenous RNase and the activity of endogenous RNase to the greatest extent. To create an RNase-free environment and strictly prevent RNase contamination is the key to successful RNA extraction.
For endogenous RNases, mainly using RNase inhibitors, currently used RNase inhibitors are:
A RNase protein inhibitor (RNasin), a protein isolated from human placenta that binds tightly to a variety of RNases to form a non-covalently bound complex that inactivates RNase. The ribozyme protein inhibitor preparation should be discarded after several freeze-thaw cycles or under oxidizing conditions. Protein inhibitors of RNase do not interfere with reverse transcription or translation of mRNA in cell-free systems;
a vanadium ribonucleoside complex, which is a complex formed by any one of vanadyl ions and four ribonucleosides, is a transition state substance, which can bind to various RNases and Highly inhibits RNase activity. However, the vanadyl ribonucleoside complex can strongly inhibit the translation of mRNA in a cell-free system, so it must be extracted multiple times with phenol containing 0.1% hydroxyquinoline to remove it;
3 diatomaceous earth, diatomaceous earth can adsorb RNase, and is removed by centrifugation during subsequent RNA purification;
4 guanidinium isothiocyanate, a powerful protein denaturant that inactivates RNase denaturation while destroying cellular structure to dissociate nucleic acids from the nucleus;
5 pyrocarbonate (DEPC), which is a strong inhibitor of RNase, but its effect is not absolute. DEPC is mainly used for RNase treatment of materials and vessels that cannot be autoclaved;
6 Other chemical reagents, such as SDS, urea, etc., also have a certain inhibitory effect on RNase.
Exogenous RNases contaminate RNA products mainly through the following ways:
1 glass products, plastic products and electrophoresis tanks;
2 pollution caused by researchers;
3 contaminated solution.
Therefore, the following measures must be taken to inhibit exogenous RNase in the experiment:
1 Laboratory glassware and plastic products are often contaminated with RNase. They must be dry baked at 180 °C for more than 3 hours (glass products) or rinsed with chloroform (plastic products) before use. Another method is to soak the glass article and other articles with 0.1% aqueous solution of diethylpyrocarbonate (DEPC) for 2 h, then rinse with sterile water several times, and dry-bake at 100 ° C for 15 min. The RNA electrophoresis tank should be washed with detergent, rinsed with water, dried with ethanol, and then immersed in a 3% H2O2 solution for 10 min, then thoroughly rinsed the electrophoresis tank with 0.1% DEPC water. Sterilized single-use plastic products are essentially RNase-free and may not require treatment;
2 In the process of RNA extraction, disposable gloves should be worn. When contacting containers that may be contaminated, gloves should be changed frequently;
3 The prepared solution should be treated with 0.1% DEPC water as much as possible at 37 ° C for more than 12 h, and then autoclaved to remove residual DEPC. The reagents which could not be autoclaved were prepared by using sterile distilled water prepared by DEPC water treatment, and then sterilized by filtration through a 0.22 μm filter.
There are various methods for preparing total RNA in eukaryotic cells, including guanidinium isothiocyanate-tantalum chloride ultracentrifugation, guanidine hydrochloride-organic solvent method, lithium chloride-urea method, hot phenol method, and guanidinium isothiocyanate method. One-step phenol-chloroform method and TRIzol reagent extraction method. At present, the common methods for extracting total RNA in the laboratory are the guanidinium isothiocyanate-phenol-chloroform one-step method and the TRIzol reagent extraction method. Preparation of total RNA from eukaryotic cells by guanidinium isothiocyanate is the combination of the most known RNase enzyme inhibitors isothiocyanate, β-mercaptoethanol and detergent N-dodecyl sarcosinate It inhibits the degradation of RNA, enhances the dissociation of nuclear protein complexes, separates RNA and proteins into solution, and selectively enters the aqueous phase free of DNA and protein, which is easily precipitated and concentrated by isopropanol.
two. Materials and Methods
1 material
Experimental fish, purchased from the market.
2 instruments, appliances
Benchtop centrifuge, constant temperature water bath (70 ° C), analytical balance, pipette, disposable syringe, square plate, tweezers, surgical scissors, petri dishes, 1.5 ml centrifuge tubes.
3 reagent
95% RNase-free ethanol; 0.1% DEPC treated water; SV RNA Lysis Buffer; SV RNA dilution Buffer; SV RNA Wash Solution; Yellow core Buffer; MnCl 2 ; 0.09M; DNase I; SV DNase Stop Solution; Nuclease-Free Water.
4 methods
1) Preparation of materials
(1) Sterilize the culture dish, scissors, and ophthalmology, and pre-cool at -20 °C.
(2) Pack the crushed ice in a foam box, place the dish on ice, and place the scissors and tweezers in the dish.
(3) Fill the bucket with crushed ice, add a small amount of tap water, and wrap the fish in gauze and put it in the bucket.
(4) Place the paralyzed fish in the square dish, cut the abdominal cavity forward and obliquely upwards with the cut anus, take out the internal organs, and separate the liver into the culture dish.
(5) Take a 1.5 ml centrifuge tube on the analytical balance and zero.
(6) Take 20-30 mg of liver tissue with a forceps in a 1.5 ml centrifuge tube and weigh.
2) Experimental operation
(1) Take about 30 mg of tissue in a 1.5 ml centrifuge tube, add 175 μl of SV RNA Lysis Buffer, crush the tissue with a disposable syringe, and mix repeatedly.
(2) Add 350 μl SV RNA Dilution Buffer (blue), turn the tube 3-4 times to mix, and set a 70 ° C water bath for 3 min.
(3) Cool on ice for 1-2 seconds, centrifuge at 14000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube.
(4) Add 200 μl of 95% ethanol and mix the tips.
(5) The above mixture was transferred to a Spin Basket Assembly, centrifuged at 14,000 rpm for 1 min, and the filtrate was discarded.
(6) 600 μl of SV RNA Wash Solution (with ethand added) was added, centrifuged at 14,000 rpm for 1 min, and the filtrate was discarded.
(7) Prepare a DNase incubation mix in a new centrifuge tube:
Yellow Core Buffer 40μl
MnCl 2 0.09M 5μl
DNase I 5μl
(8) The above 50 μl mixture was directly applied to the spin basket membrane and incubated at room temperature (20-25 ° C) for 15 min.
(9) Add 200 μl of SV DNase Stop Solution (with ethand added) to Spin Basket 14000 rpm and centrifuge for 1 min.
(10) Add 600 μl of SV RNA Wash Solution, centrifuge at 14,000 rpm for 1 min, and discard the filtrate.
Yellow core Buffer 40μl MnCl2, 0.09M 5μl DNase I 5μl
(11) Add 250 μl of SV RNA Wash Solution, centrifuge at 14,000 rpm for 2 min, and transfer the Spin Basket to a new centrifuge tube.
(12) Add 50 μl of Nuclease-Free Water to the membrane, centrifuge at 14000 rpm for 1 min, dissolve the RNA, and store at -20 °C.
(13) Take 5 μl of RNA sample and measure the RNA quality by 1% agarose gel electrophoresis.
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