I am trying to extract RNA from primary culture using Trizol. But after checking on the bioanalyzer I obtained very low RIN value (3.4) and the electropherogram showed no peaks (only the marker peak is visible). I am assuming even if there is degradation, I should still be able to see small degradation peaks although not 18s and 28s. But the graph does not show anything. Nanodrop showed a concentration of around 200ng/ul. So there should be still some RNA in the sample, but I am unable to understand, why there are no peaks observed? I am wondering where did all the RNA go or is it degraded very badly and no peaks can be seen? Also, Trizol inhibits RNAse so what are the other sources of RNA degradation?
First of all I think you have to make sure your Bioanalyzer works very well and try to set it correctly. Second, I think your RNA is very low, and that is why its being detected. Try to repeat your work ideally and check again. If it still same problem, then I think the problem might related to your cells.
Good luck!
Dear Renuka
I believe you need to repeat the RNA isolation process. There may be some error in your process of isolation which you may have skipped in the first time. Although Trizol degrade RNA, it stay only for a very small time during initial stage and therefore, i do not think it could e degrading your sample. If the problem persist, then you may re calibrate your bio analyzer.
Hope this helps
Hi Renuka,
You may need to repeat the isolation. Check your NanoDrop 260/280 and 260/230 ratios which should tell you if there are any contamination issues. In ideal conditions these ratios should be 2.0 and 2-2.2 respectively. Your EPG shows a signal around 6k nt which might be your RNA signal, in that case your sample is highly degraded.
Also make sure you followed your sample prep for TapeStation analysis properly, make sure to mix your sample and buffer properly, the heating and cooling step, use RNase free consumables (during analysis and extraction) and clean work bench. The marker has run properly indicating the instrument conditions are fine.
Good luck!
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