RNA quality testing and identification

Method 1: Detect the absorbance of RNA solution

The absorbances at 280, 320, 230, and 260 nm represent the values ​​of nucleic acids, background (solution turbidity), salt concentration, and protein. In general, we only look at OD260 / OD280 (Ratio, R). At 1.82.0, we think that the contamination of protein or other organic matter in RNA can be tolerated, but please note that when you use Tris as a buffer to detect absorbance, the R value may be greater than 2 (generally should be <2.2) . When R <1.8, the contamination of protein or other organic matter in the solution is more obvious. You can decide the fate of this RNA according to your needs. When R> 2.2, it means that the RNA has been hydrolyzed into a single nucleic acid.

Method 2: RNA electrophoresis

In general, RNA electrophoresis is performed with denatured gel, but according to my experience, if you only want to check the quality of RNA, it is not necessary to conduct such a troublesome experiment, use ordinary agarose gel. The purpose of electrophoresis is to detect the integrity of 28S and 18S bands and their ratio, or the integrity of mRNA smear. In general, if the 28S and 18S bands are bright, clear, and sharp (meaning the edges of the band are clear), and the brightness of the 28S is more than twice the 18S band, we believe that the quality of the RNA is good. The above are the two methods we commonly use, but neither of these methods can clearly tell us whether there is residual RNase in the RNA solution. If there is a very small amount of RNase in the solution, it is difficult to detect by the above method, but most of the subsequent enzymatic reactions are above 37 degrees and are carried out for a long time. In this way, if there is a very small amount of RNase in the RNA solution, there will be a very suitable environment and time to play their role in the subsequent experiment, of course, your experiment will be over. Below, we introduce a method that can confirm whether there is residual RNase in the RNA solution.

Method 3: Insulation test

The method is very simple, according to the sample concentration, draw two 1000 ng RNA from the RNA solution into a 0.5 ml centrifuge tube, and make up to a total volume of 10 ul with pH 7.0 Tris buffer, then close the tube cap . Place one portion in a constant temperature water bath at 70 ° C and keep warm for 1 h. Place the other in a refrigerator at -20 ° C for 1 h. After the time is up, two samples are taken for electrophoresis. After electrophoresis is complete, compare the electrophoretic bands of the two. If the bands of the two are the same or there is no obvious difference (of course, their bands must also meet the conditions in Method 2), it means that there is no residual RNase contamination in the RNA solution, and the quality of the RNA is very good. Conversely, if the samples incubated at 70 ° C degrade significantly, it means that the RNA solution is contaminated with RNase.