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I'm trying to set up Real-time PCR analysis (relative quantification) in adipocyte tissue (biopsies) and
this in 2 depots (visceral and subcutaneous). I tested some primers by making
standard curves with 5 1/5 dilutions (from a pool cDNA), starting with '100' as
my non-diluted cDNA (10 ng/ul). For all my standard curves I remarked that the
'100' completely falls out of the curves, so my efficiency is very low (see attachment).
By omitting the '100' I have excellent standard curves. I'm thinking that, because
of working with fatbiopsies, that I have a lot of inhibitors in my samples. So
by diluting the samples, I also dilute the inhibitors which increase my
efficiency.
Therefore, I decided to dilute my samples 1/20 for doing the RT-PCR analysis.
This means that I have only 0,5 ug/ul cDNA for each sample (I use 4 ul cDNA in
the reaction mix). For my potential HKG I have Ct values around 20-24 but some
of my GOI have Ct values >30. I'm not able to isolate more RNA from my
fatbiopsies.
Is 0,5 ug/ul cDNA enough for doing reliable RT-PCR (total of 2ug/well)? And if
so, can I trust on Ct values >30 if they are reproducible? If not, would it be a
solution to do a nested RT-PCR reaction (take the amplified cDNA of a previous
reaction) for the GOI which have high Ct-values?
Many thanks,
Helene
Attached Thumbnails
Edited by hdnaeyer, 09 July 2009 - 01:08 AM.
