Is a slightly superior to 0.5 (range: 0.6 to 0.9) 18S:28S rRNA ratio (on a formaldehyde denaturing agarose gel) OK to conclude that my total RNAs are intact enough for RT-qPCR? I do not have access to a Bioanalyzer, alas.
Total RNA have been extracted from deep frozen liver with the NucleoSpin RNA kit under reasonably good RNase-free conditions (I think).
Here's my gel (we don't have a RNA ladder in the lab...):
(Thanks a lot !)
Hi Julien. Your rRNA sub units are still distinct - even though some degradation is obvious - so yes I would you could use that RNA for qPCR.
I would try and situate my primers though within 1kb if your mRNA 3' terminus to ameliorate any distortions linked to RNA degradation affecting message representation
Thanks a lot, I'm quite reassured.
Without any degradation, my lanes would display only 2 sharp bands (18S and 28S rRNA), am I correct?
(I'll try to redesign the primer pairs if it's possible)
Your RNA actually looks very good - in agreement with Dr. Hall above. The other striated patterns you see are indeed the other mRNA species of different lengths - perfectly normal - and actually nice to see.
Thanks for your qualification Jack, i.e. the distinction between bona fide degradation and variance due to simple mRNA lengths
The cDNAs I've synthesized gave us quite good results in the RT-qPCR experiment that followed.
Thank you very much Jack (and Laurence) !
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