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Degenerated qPCR data after keeping the cDNA in the fridge. - (May/22/2007 )


I make the cDNA and dilute it 5 times with 0.5 x TE buffer and keep it in the fridge at 4 C. The cDNa was made at 37C and on ice,
no 90 C step in the end. I do the qPCR the following days and I see that the data ie. sham group and control group in each gene is more and more
NOT the same and the treated groups are all over the place.

Could it be because the cDNA is sticking to the plastic?
Could it be because I whirlemix the RT enzyme - RNA mix?




I think it may be problematic that you did not inactivate the RT I think this is an important step to keep the nuclease activity from messing up your sample...

Please don't store your cDNA at 4C it should be made into aliquots and put at -20 -- it wont take that long to thaw...



I completely agree with beccaf22 - if you don't inactivate the RT PLUS you keep the cDNA in the fridge (where the RT is still active) you are very likely running into problems. Depending on the RT you have some endonuclease capacity - voila - there goes your cDNA...