I extracted total RNA from frozen PBMCs using Norgen's total RNA extraction kit so that I could retain microRNAs for a small RNA sequencing experiment. The tapestation trace is bizarre! The 28S/18S ratios are really high (to the point of being unbelievable) and the RINe values are awful.
I wonder whether the retention of small RNAs interferes with the tapestation's estimate of RINe given that RINe "represents the relative ratio of the fast zone, the region between the 18s signal peak and the small RNAs".
As I understand it degraded RNA is less of a problem in small RNA library preps compared with other preps (e.g. poly A selection and ribodepletion) so perhaps it doesn't matter, nevertheless I am bemused by the traces and wondered if anyone could explain them. I have attached the traces to illustrate my point.
In my opinion large black band is degradaded DNA and at the botton 5SRNA. In your gel i do not see 28/18S. Have you treated your sample with DNAase-I ?
No, the kit said it was good at removing gDNA so I haven't removed it with DNAse but having spent the evening looking at traces I think you may well be right. I will try added in a DNAse step.
In fact it is quite difficult to obtain distinct 28/18S from the frozen blood, if only it wasn't frozen at -70.
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