RNA extraction technology is not only an important part of molecular biology technology, but also an important foundation of functional genomics research technology. Studying the regulation mechanism of genes in organisms from the RNA level has become an important means of molecular biology research. To conduct character research on a certain organism or tissue, the first step is to obtain the character gene. Isolation of complete RNA from tissue cells is essential for molecular cloning and gene expression analysis, such as Northern blot and hybridization analysis, cDNA synthesis, etc. The effectiveness of the experiment largely depends on the quality of the RNA. Using the extracted RNA, people can quantitatively detect specific gene expression and accurately understand the laws of cell life activities from the molecular level. Usually a typical mammalian cell contains about 10-5μg RNA, of which 80%-85% is rRNA (mainly 28S, 18S, 5.8S, and 5S); 10%-15% is tRNA and small nuclear 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). On the contrary, 1% to 5% of total RNA is mRNA. Although mRNAs vary in size and nucleotide sequence, ranging from hundreds to thousands of bases, most eukaryotic mRNAs have an oligoadenylic acid (polyA) tail at their 3'end. The length is generally sufficient to adsorb to oligodeoxythymidylate-cellulose [oligo(dT)] so that mRNA can be separated by affinity chromatography. This population encodes all the polypeptides synthesized by the cell. Studies have shown that RNA is extremely unstable and easy to degrade, while RNAse is almost everywhere and is particularly stable. Therefore, the key factor when extracting RNA is to avoid contamination of exogenous RNAse and inhibit 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, RNase inhibitors are mainly used. Currently, the commonly used RNase inhibitors are:
① Protein inhibitor of RNase (RNasin), which is a protein isolated from human placenta, can be closely combined with a variety of RNases to form a non-covalently bound complex to inactivate RNase. RNase protein inhibitor products should be discarded after freezing and thawing several times or under oxidizing conditions. Protein inhibitors of RNase do not interfere with reverse transcription or translation of mRNA in a cell-free system;
②Vanadox ribonucleoside complex, which is a complex formed by vanadyl ions and any one of the four ribonucleosides. It is a transition state analogue that can combine with a variety of RNases. Efficiently inhibit RNase activity. However, the vanadyl ribonucleoside complex can strongly inhibit the translation of mRNA in a cell-free system, so it must be extracted several times with phenol containing 0.1% hydroxyquinoline to remove it;
③Diatomite, diatomite can adsorb RNase and remove it by centrifugation in the subsequent RNA purification process;
④ Guanidine isothiocyanate, it is a powerful protein denaturant, which destroys the cell structure and dissociates the nucleic acid from the nucleus at the same time also denatures and inactivates the RNase;
⑤Diethyl pyrocarbonate (DEPC), it is a strong inhibitor of RNase, but its effect is not absolute. DEPC is mainly used for RNase treatment of materials and utensils that cannot be autoclaved;
⑥ Other chemical reagents, such as SDS, urea, etc. also have a certain inhibitory effect on RNase.
Exogenous RNases mainly contaminate RNA products through the following ways:
①Glass 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:
①The common glass products and plastic products used in the laboratory are often contaminated with RNase. They must be dry-baked at 180℃ for more than 3 hours (glass products) or rinsed with chloroform (plastic products) before use. Another method is to soak glass products and other articles with 0.1% diethyl pyrocarbonate (DEPC) aqueous solution for 2 hours, then rinse them with sterilized water several times, and dry-bake them at 100°C for 15 minutes. The RNA electrophoresis tank needs to be washed with detergent, rinsed with water, dried in ethanol, soaked in 3% H2O2 solution for 10 minutes, and then thoroughly rinsed with 0.1% DEPC water. Sterilized single-use plastic products are basically RNase-free and do not require treatment;
②In the process of RNA extraction, disposable gloves should be worn, and gloves should be changed frequently when touching potentially contaminated utensils;
③The prepared solution should be treated with 0.1% DEPC water at 37℃ for more than 12h as much as possible, and then autoclaved to remove residual DEPC. For reagents that cannot be autoclaved, use sterile distilled water that has been prepared with DEPC water, and then filter and sterilize with a 0.22μm filter.
There are many methods for preparing total RNA from eukaryotic cells, including guanidine isothiocyanate-cesium chloride ultracentrifugation method, guanidine hydrochloride-organic solvent method, lithium chloride-urea method, thermal phenol method, guanidine isothiocyanate method— Phenol-chloroform one-step method and TRIzol reagent extraction method. At present, the commonly used methods for extracting total RNA in laboratories are guanidine isothiocyanate-phenol-chloroform one-step method and TRIzol reagent extraction method. The guanidine isothiocyanate method to prepare eukaryotic total RNA is a combination of the strongest known RNase inhibitor guanidine isothiocyanate, β-mercaptoethanol and the detergent N-sarcosyl sodium It inhibits the degradation of RNA, enhances the dissociation of nucleoprotein complexes, separates RNA and protein and enters the solution, RNA selectively enters the water phase without DNA and protein, and is easily concentrated by isopropanol precipitation.
二、Materials and Methods
Experimental fish, purchased from the market.
2、Instruments and utensils
Benchtop centrifuge, constant temperature water bath (70℃), analytical balance, pipette, disposable syringe, square plate, tweezers, surgical scissors, petri dish, 1.5ml centrifuge tube.
95% RNase-free ethanol; 0.1% DEPC treated water; SV RNA Lysis Buffer; SV RNA dilution Buffer; SV RNA Wash Solution; Yellow core Buffer; MnCl2; 0.09M; DNase Ⅰ; SV DNase Stop Solution; Nuclease-Free water .
1) Preparation of materials
(1) Sterilize the petri dish, scissors, and ophthalmic forceps, and pre-cool at -20°C.
(2) Use a foam box to contain crushed ice, place the petri dish on ice, and place the scissors and tweezers in the petri dish.
(3) Fill the bucket with crushed ice, add a small amount of tap water, wrap the fish with gauze and put it in the bucket.
(4) Place the paralyzed fish in a square plate, cut the abdominal cavity forward and obliquely upward with scissors, take out the internal organs, separate the liver and place it in a petri dish.
(5) Take a 1.5ml centrifuge tube on the analytical balance and adjust the zero.
(6) Use forceps to take 20-30mg of liver tissue into a 1.5ml centrifuge tube and weigh.
2) Experimental operation
(1) Take about 30mg of tissue in a 1.5ml centrifuge tube, add 175μl of SV RNA Lysis Buffer, crush the tissue with a disposable syringe, repeatedly beat and mix.
(2) Add 350μl SV RNA Dilution Buffer (blue), turn the tube 3-4 times to mix, and place in a 70°C water bath for 3 minutes.
(3) Cool on ice for 1-2 seconds, centrifuge at 14000 rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube.
(4) Add 200μl of 95% ethanol and mix with pipette tip.
(5) Transfer the above mixture into Spin Basket Assembly, centrifuge at 14000 rpm for 1 min, and discard the filtrate.
(6) Add 600μl SV RNA Wash Solution (with ethand added), centrifuge at 14000rpm for 1min, discard the filtrate.
(7) Prepare DNase incubation mix in a new centrifuge tube:
Yellow Core Buffer 40μl
MnCl2 0.09M 5μl
DNase Ⅰ 5μl
(8) Add the above 50μl mixture directly to the spin basket membrane and incubate at room temperature (20-25°C) for 15 minutes.
(9) Add 200μl SV DNase Stop Solution (with ethand added) to Spin Basket 14000rpm, and centrifuge for 1min.
(10) Add 600μl SV RNA Wash Solution, centrifuge at 14000rpm for 1min, discard the filtrate.
Yellow core Buffer 40μl MnCl2, 0.09M 5μl DNase I 5μl
(11) Add 250μl of SV RNA Wash Solution, centrifuge at 14000rpm for 2min, and transfer the Spin Basket to a new centrifuge tube.
(12) Add 50μl Nuclease-Free Water to the membrane, centrifuge at 14000rpm for 1min, dissolve RNA, and store at -20°C.
(13) Take 5μl of RNA sample and check the quality of RNA by 1% agarose gel electrophoresis.