Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

qPCR in primary cells - housekeeping CT-values sample-specific bad - (Nov/14/2011 )

Hello everyone,

I've isolated RNA from cells, which were stored very long-time (up to >10-15 years) in Carnoy solution. I measured it on a nanodrop and did everything as usual.
Now I have extremely high CT-values for my housekeeping gene in several of these samples. Could this be related to the poor sample quality and can anyone tell me up to which CT-values it would be ok to use the expression data?

Thank you very much!!



Carnoy's solution should be fixing the RNA a bit, but 10 years is way to much IMHO. I'm not sure about the process of fixation, so your RNA can be either degraded of just fragmented like in formaline, both leading to high Cts.

Usually it's not good to use Cts higher than 30 for expression. You will see yourself that near the 30 the SD of replicates becomes very high. If you are looking for a very abundant gene, you may be able to do expression studies by choosing housekeeping gene, that is also very abundant. But if your target gene is also above 30, that's not generally quantifiable.