Hi everybody,
I would like to assess the quality of my RNA before performing further analyses (RT-qPCR) but I do not have access to Bioanalyzers like the Experion or Agilent 2100 Bioanalyzer. I know I could run my sample on an agarose gel, but I heard that there are genes whose RNA gets degraded earlier compared to the others and I was wondering if there is a way to use them as marker for RNA degradation.
Hopefully this does not sound too silly of a question!
Thanks in advance
Hi Nicolò,
It's not exactly the answer to your question, but there is this paper from Fleige & Pfaffl (2006) that can be helpfull :Article RNA integrity and the effect on the real-time qRT-PCR performance
Good luck ,
Hi Nicolò,
the RNA quality can be a source of bias for quantification even for small qPCR products depending on the RT priming strategy (polydT) and the location of the amplicon in relation to the 3´-end (starting point of RT). An easy possibility to account for differences between samples is to measure two amplicons targeting the same transcript but on different locations (i.e. 5´and 3´ end) of a housekeeping gene. The ratio tells you something about the RNA quality (and length of RT processivity). We use 5´and 3´ GAPDH amplicons and see nice correlations with the RIN number of used RNA. For random primed RT there should not be a large bias in qPCR data as mentioned by Matthias. Concerning your question about the possible different stability of RNA species I would differentiate between endogenous stability (this varies indeed between transcripts) and "exogenous degradation". I would expect that there is no or only little difference in stability related to exogenous degradation (during RNA prep etc) between RNA species.
Good luck
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