I extracted total RNA from fungal samples and my goal is to send the RNA for RNAseq, however my RNA did not pass the quality control due to atypical baseline by the bioanalyser results (Please see image attached).
The total RNA was extracted using the QIAGEN RNeasy mini kit and DNase treatment was done after RNA extraction using the PROMEGA RQ1 RNase-free DNase.
Does anybody know why I am getting this atypical RNA quality results? Thank you very much!
Hello,
it seems some degradation/contaminants are associated with your samples. In case you cannot rerun the exp then I would recommend doing a second RNA extraction with your RNA sample. The total yield will decrease but you should have a better quality RNA for downstream applications.
good luck.
F.
Thank you very I much for your insights Dr. Fathi Karouia. At first I ruled out the RNA degradation because of the good clean bands when running a denaturing gel, and RNA quantity with Qubit was really good. So I suspected that the atypical baseline was due to contamination. I did perform the experiment a second time for a new RNA extraction. However, instead of using the PROMEGA DNase after the RNA extraction I used a QIAGEN DNase compatible with the RNeasy mini kit for a in-column DNA digestion. It worked perfectly and I got a high quality RNA this way. So I am curious to see if any other researcher ever experienced problems with DNase treatment after RNA extraction for downstream applications?
Good job. My experience suggests that doing in column DNA treatment work best with yield and quality. Maybe it is also a combination of mixing qiagen and promega protocols. However, I recall your DNA treatment was done after the RNA extraction. So, it should be ok. Anyway, you did troubleshoot so you should be happy about yourself. Keep the good work.
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