Three critical steps, if not performed well can cause RNA degradation. Handling and storing of samples, disruption of samples and storage of eluted RNA. (1) Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissecting. If samples cannot be processed immediately, it should be flash frozen in liquid nitrogen and stored at -80°C. Samples should be handled with RNase-free tools. (2) When the sample is disrupted, disruption needs to be fast and thorough. Slow disruption (e.g. placing cells or tissue in RB Buffer without any additional physical shearing) may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in the buffer. (3) After elution of RNA with RNase-free ddH2O, store RNA at -80°C. (4) Degradation of RNA may also occur during loading into a gel. Use gel and fresh running buffer prepared using DEPC-treated ddH2O, as well as a properly cleaned geltray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining.
Why does the obtained RNA appear smeared and degraded? "