pcrman, on 05 June 2011 - 02:34 PM, said:
Despite the pattern is different from what is shown in the gel (which is denaturing agarose gel) from Ambion site, there is no question that your RNA is degraded. You may also have DNA contamination in your RNA. Degradation of RNA (which is time and temperature dependent) during agarose gel run may not be a big concern, but still you should clean the comb and gel box with Rnase zap and RNase free reagents. The ability of amplifying highly expressed genes such as GAPDH doesn't exclude RNA degradation. When RNA is degraded partially, you may still be able to amplify highly expressed genes, but need more cycles.
Thanks. I used 1 ml of TRIzol for 107
cells, which is the limit according to the recommended protocol. If there is DNA contamination, do you think that is the reason? And I used -80 frozen cells, instead of fresh cells. Does it matter?
If GAPDH couldnt work, how about other non-housekeeping genes?
How should I test if the RNA is degraded or not? Even if it is degraded, the OD value is not affected, is that right? Is RNA electrophoresis the only way? Thank you very much.