It has always been a hassle to obtain pure RNA from tissue or cultured cells. RNA is frequently degraded by ubiquitous RNases which are hard to get rid of. As autoclaving won’t inactivate RNases, all buffers involved in RNA extraction have to be treated with diethylpyrocarbonate (DEPC) and glassware has to be baked overnight. Another problem is in isolating RNA is contamination by DNA. Nevertheless, the purity and integrity of RNA is crucial for its effective use in downstream applications, such as reverse transcription (RT-PCR), Northern blotting, RNase protection assays, in vitro translation and many more.
All solutions in Promega’s SV Total RNA Isolation System come RNase-free, including the spin column assemblies and elution tubes. Other than a 95% RNase-free ethanol solution, there is no need to prepare your own buffers and plastic materials. The guanidine thiocyanate-containing RNA Lysis Buffer has to be supplemented with beta-mercaptoethanol (provided), which inactivates RNases present in all cell extracts. The solution is then refrigarated at 4°C. The rehydrated DNase I should be kept frozen at -20°C. All other solutions can be stored at room temperature and the isolation itself is accomplished at room temperature, too.
The kit was originally designed for RNA isolation from leukocytes, plant tissues, bacteria, yeast and adherent culture cells. In addition to RNA extraction from MRC5 cells, I used this kit to extract viral RNA from cell culture supernatant. The kit worked very well in both extractions. The kit can be used to isolate genomic DNA, too.
The columns are compatible with a microcentrifuge or a vacuum manifold. I have only used the spin protocol. I extract RNA from approximately 106 cells per purification. After lysis of cells, the sample is diluted with SV RNA dilution buffer and ethanol. The sample is then washed and treated with prepared DNase I solution. I have found that incubation with DNase should take place for at least 25 minutes; the protocol suggests15 minutes. After addition of DNase Stop solution and 2 washing steps, the total RNA is eluted in nuclease-free water. Yields were good as were 260/280 ratios: Total RNA yield was 50-100 µg; A260/280 ratios were between 1.9 and 2.1.
The eluted RNA was of great quality, intact and stable, which could be shown in downstream experiments. I used the eluted pure RNA in several RT-PCR assays, all of which worked very well.
Apart from the fact that there are a lot of different solutions to be used (8 provided), which can be a bit confusing, RNA isolation with this kit is straight-forward and fast. The whole protocol takes only 1-1.5 hours, without disagreeable phenol/chloroform extractions or ethanol precipitations.