I'm having problems with qpcr. This primer have been tested in another lab and it worked fine. It has late amplification (like 30 cycles) but in my samples I get amplification in like 45 cycles. I'm doing rna extraction with trizol and I'm doing retrotranscription with superscript II and oligo dT (using 2,5 ug rna). Gapdh qpcr works just fine. Any suggestions?
Yes, but i need to measure relative expression. Does it work if I use the qpcr primer in the Retrotranscription?
If you are using exactly the same conditions as the other lab (same amount of starting cDNA, same type of sample and same techniques) maybe there is a problem with the integrity of the RNA on your samples.
From what i´ve seen, when working with tumors (fresh and ffpe), RNA samples obtained from ffpe tissuee will require more starting material in order to amplify somewhere around the same cycle in which you will normally notice amplification in the fresh tissues.
Hope this helps
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