ISH vs. Real Time PCR - correlation between CT-values and quantity - (Nov/27/2007 )
I have a really basic question to all of you concerned with IHS and Real Time PCR. I am working on the estimation of cell cycle markers (cyclins and cyclin-dependent-kinases) in feline neurons - and I have yet failed to detect their mRNAs by using ISH in formalin-fixed-paraffin-embedded tissue sections - no signals at all. So we decided to verify if there is any expression of those markers by using Real Time PCR - and for some we got some curves, but at very high CT-values (cycle 35 and higher).
So is there any common correlation between the CT-values and the possibility to detect them in the tissue sections by ISH- e.g. could it be said in general "if you have CT-values higher then 30 (for instance), the mRNA-quantity is under the detection limit of ISH"?
I hope you can give me some hints,
best regards, Barbara
If you do the experiment you can certainly say that for your samples... but maybe there is something else wrong with the ISH? what is the standard limit of detection for this? (I am not that familiar with hybridization but isn't the signal amplified by multiple products being converted by the same enzyme?) Is RNA stabilized by this fixation or could there be degradation? Also be careful because there may be false positives in PCR at very high Ct values, be sure to confirm say check on a gel, ensure water controls do not amplify at high Ct values etc. Would you expect that neurons actually do not have mRNA for these cell cycle markers because they dont normally divide right?
it sounds like there isn't any amplification of the product... it sounds like primer dimer being amplified there.
if it were product, you'd see amplification a loot sooner than 35.
how does the melt curve look on the RT PCR?