SV Total RNA Isolation System From Promega

The SV Total RNA Isolation System was designed to purify intact total RNA from tissues, cultured cells and white blood cells with either a spin or vacuum (SV) purification method. Up to 60 mg of tissue can be processed per purification, depending on the type, function and RNA expression levels of the tissue. DNase treatment is included to prevent genomic DNA contamination, which can interfere with further applications. RNA extraction is achieved without the use of phenol:chloroform extractions or ethanol precipitations, and there is no DNase present in the final RNA preparation. The SV Total RNA Isolation System can also be used to isolate both genomic DNA and RNA from the same sample, but I have always used it only for total RNA isolation. I have isolated RNA from several different sample types, including Xenopus embryos, mouse tissues (lung, heart) and human peripheral blood monocytes.

Before starting, four solutions must be prepared: RNA Lysis Solution (RLS), RNA Wash Solution (RWS), DNase Stop Solution (DSS), and the DNaseI must be reconstituted in sterile double-distilled water and aliquoted. Although it is possible to freeze-thaw DNaseI, we found that is better to make many small aliquots and use DNase only once.

The RNA Lysis Solution (RLS) combines the disruptive and protective properties of guanidine thiocyanate (GTC) and β-mercaptoethanol to inactivate the ribonucleases present in cell extracts. GTC, in association with SDS, disrupts nucleoprotein complexes, allowing the RNA to be released into solution, free of proteins. It is very important to thoroughly disrupt the tissue or cell pellet at this stage. Any non-lysed particles will decrease the final RNA yield. It is not necessary to transfer the cell lysate into a fresh tube; you can simply add required amount of the Dilution Solution into the same tube and proceed to centrifugation. This reduces the total procedure time and decreases the risk of loosing RNA. The Dilution Solution causes selective precipitation of cellular proteins, while the RNA remains in solution. After centrifugation to clear the lysate of precipitated proteins and cellular debris, the RNA-containing supernatant is then applied to the spin column. Each spin column sits in a 2 ml collection tube. The RNA binds to the silica surface of the glass fibers in the spin column. Following centrifugation (or vacuum filtration), RNase-Free DNase I is applied directly to the silica membrane to digest contaminating genomic DNA. The prepared DNase is yellow which makes it very easy to ensure that it covers the entire membrane. After a 15 minute incubation with the DNase, the bound total RNA is further purified from contaminating salts, proteins and cellular impurities by washing. Finally, the total RNA is eluted from the membrane by the addition of Nuclease-Free Water. In order to elute more concentrated RNA, it is possible to elute it in as little as 30 µl heated water. Test the RNA concentration by spectrophotometer. Unfortunately, sometimes it happens that RNA is not eluted from the column. I check my eluate with a spec. If I find little or no RNA, I re-elute with double-distilled sterile water heated to 65ºC and re-check RNA on a spec. You can also put the column into 65°C waterbath for about 10-15 minutes, then repeat the elution step.