In order to obtain high-quality eukaryotic cell mRNA, RNase inhibitors must be used or the following method of simultaneous cell disruption and inactivation of RNase must be used to minimize the activity of RNase released during cell disruption. At the same time, it is also important to avoid accidental introduction of other potential trace RNases in the laboratory. Here are some precautions to avoid RNase staining. Most experienced researchers do not stick to these precautions, but may use one or several of them to solve problems when they encounter problems.
Method for simultaneous cell disruption and inactivation of RNase
(1) Experimental procedures
If not handled carefully, exogenous RNases can contaminate RNA products through the following ways:
(1) Glass products, plastic products and electrophoresis tanks Sterilized disposable plastic products are basically RNase-free, and can be used directly to prepare and store RNA without pretreatment. Ordinary glassware and plastic products used in the laboratory are often stained with RNase, and must be dry-baked at 180 ℃ for 8 hours or more (glassware) or rinsed with chloroform (plastic products) before use. Another method is to soak the beakers, test tubes and other supplies used to prepare RNA with an aqueous solution of 0.1% diethyl pyrocarbonate (DEPC). DEPC is a strong inhibitor of RNase, but its effect is not absolute (Fedorcsak and Ehrenberg, 1966). The glass or plasticware filled with DEPC was placed at 37°C for 2 hours, then rinsed several times with sterile water, and dried at 100°C for 15 minutes (Kumar and Lindberg, 1972). Autoclave at 15 lbf/in2 (1.034x105Pa) with chlorine for 15 minutes. The above treatment can remove the trace amount of DEPC on the armour to prevent DEPC from modifying the purine base of RNA through carboxymethylation.
Carboxymethylated RNA has low translation efficiency in a cell-free system. However, unless most of its purine bases are modified, its ability to form DNA:RNA or RNA:RNA hybrids is not significantly reduced. The electrophoresis tank for RNA electrophoresis should be cleaned with detergent, rinsed with water, dried with ethanol, then filled with 3% H2O2 solution, placed at room temperature for 10 minutes, and then rinsed thoroughly with 0.1% DEPC-treated water . It is best to set aside some glassware, plastic products and electrophoresis tanks for special marks, and store them in designated places for RNA experiments.
(2) Pollution caused by researchers The main potential source of RNase pollution is the hands of researchers. Therefore, when preparing materials and solutions for isolation and analysis of RNA, you should wear disposable gloves during all operations involving RNA. After touching "fat" glassware and other objects, the gloves may be contaminated with RNA. Enzymes, so you should change gloves frequently when performing RNA experiments.
(3) Contaminated solution Prepare the solution with autoclaved water and chemical reagents for RNA research, weigh the reagent with a dry roasted spoon, and put the solution into RNase-free glassware. If possible, the solution should be treated with 0.1% DEPC at 37°C for at least 12 hours, and then heated at 100°C for 15 minutes or steam sterilized under high pressure of 15lbf/in2 (1.034x105Pa) for 15 minutes. Note: DEPC can react quickly with amines, so it cannot be used to process buffers containing Tris. Several bottles of new unopened Tris crystals can be stored to prepare an RNase-free solution.
(2) Inhibitors of RNase
Here are three widely used specific RNase inhibitors.
(1) The protein inhibitor of RNase is a protein isolated from human placenta that can bind tightly with a variety of RNases (KI≈3x1010) to form a non-covalently bound equimolar complex to inactivate RNase. This protein may be an inhibitor of angiogenin in vivo. Angiopoietin is an angiogenic factor whose amino acid sequence and predicted tertiary structure are similar to pancreatic RNase. Several manufacturers sell this inhibitor under different trade names. The protein should be placed in 50% glycerol containing 5mmol/L dithiothreitol (DTT) and stored at -20°C. The inhibitor product should be discarded after being frozen and thawed several times or placed under oxidizing conditions, because the above treatment will denature the protein and release the bound RNase. Therefore, this protein inhibitor should not be used in the initial step of RNA extraction using denaturants to lyse mammalian cells. However, this inhibitor should be used when using a more gentle lysis method, and the protein should be present in all subsequent RNA purification steps. Since phenol extraction can remove protein inhibitors, inhibitors should be added several times during the purification process. Its maximum activity requires a sulfhydryl reagent, and it does not interfere with reverse transcription or mRNA translation in a cell-free system.
(2) Vanadyl ribonucleoside complex. This complex formed by vanadyl (1V) ion and any one of 4 kinds of ribonucleosides is a kind of transition state analogue, which can interact with many kinds of RNase binding and several branches can inhibit the activity of RNase 100%. These four vanadyl ribonucleoside complexes can be added to intact cells, and the concentration used in all processes of RNA extraction and purification is 10mmol/L. The resulting mRNA can be directly translated in silicon oocytes and can be used as a template for some external enzymatic reactions (such as mRNA reverse transcription). However, the vanadyl ribonucleoside complex strongly inhibits the translation of mRNA in a cell-free system, so it must be extracted with phenol containing 0.1% hydroxyquinolin [equilibrated with 0.01 mol/LTris.Cl (pH 7.8)] To remove it. Several companies sell vanadyl ribonucleoside complexes.
(3) Macaloid (on the diatom) Macaloid is a kind of clay. It was discovered many years ago that it can adsorb RNase, and it is made into a slurry with a buffer solution to dissolve the cells at a final concentration of 0.015% (W/V). This clay along with the RNase adsorbed by it can be removed by centrifugation in the subsequent RNA purification process (such as after phenol extraction).
(3) The method of simultaneous cell disruption and RNase inactivation
Strong denaturation such as guanidine hydrochloride or guanidine thiocyanate solution can quickly dissolve the protein, causing the cell structure to break, and the nucleoprotein is dissociated from the nucleic acid due to the destruction of its secondary structure. RNase can withstand a variety of treatments, so the above reagents can be combined to extract intact RNA from tissues, such as the pancreas rich in RNase. The following series of experimental procedures use RNase inhibitors and/or related methods that can quickly inactivate RNase to isolate total RNA, nuclear RNA, and cytoplasmic RNA from tissue or cell culture.