RT-PCR Dose Dependence?

cnmcomstock cnmcomstock at insightbb.com
Thu Apr 15 18:06:56 EST 2004


Hello;

 I have been trying to quantify a few genes by RT-PCR.  My control reactions
show nice dose dependence (Ct values from 20-25) with an efficiency about
85%.  However, the genes I'm interested in have Ct values around 30 and do
not show any dose dependence from an estimated 50-1000ng cDNA (non-template
control Ct 35-40).  My melt curves often yield sharp product peaks in the
high 80's, the NTC show broad peaks in the low 80's.  I have used multiple
sets of primers (currently up to 13 sets) and they all end with the same
result.

Any comments or suggested literature would be greatly appreciated.  Thanks
Clay





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