Hi! I am new to RT-qPCR and I have the following question.
We are planning to measure some miRs in plasma in a longitudinal cohort. As far as I know, there aren't any generally accepted reference genes to use as endogenous controls for plasma.
We are thinking using absolute quantification with synthetic miR standards and report results as copies/ul of plasma.
If we use AQ, do we still need to normalise against eg. cel-miR-39, to account for variability in extraction? What would be the best method for normalisation in this case? Thank you in advance!
Can you please let me know which kit you are using for miRNA RT-qPCR?
For this study we are planning to use either Qiagen miRNeasy either mirVana PARIS for extraction (we have a working protocol for both and final selection will depend on cost quotations, because the samples are too many) For RT-qPCR we use Taqman.
Thank you very much for your reply. Any help is welcome.
I will suggest you to try with RUN48 as endogenous control for RT-qPCR. We worked with this and is fine with human samples. You will get the universal primer from kit that you will use for cDNA conversion.
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