Hi everyone,
my colleague is using the Taqman assays for his gene expression analysis. After running his assay with a standard curve it looks like as if his primers have an efficiency of 170%.
Does anyone have an idea, what could be causing this?
I usually work with Sybr green and first thought of unspecific amplification but clearly this should not happen with the Taqman assay, I would have thought.
Thank you in advance for your help.
Hi,
Poor cDNA synthesis, dilution issues, DNA contamination, carry over of PCR inhibitors, pipetting errors, RNase contamination or etc. He/she must double check everything.
Good luck.
Hi Joanna, apart from pipetting, dilution and template irregularities, also the presence of excessive primer dimers induces high efficiency, and often if the dynamic range of your standard curve is not enough this may as well lead to the efficiency above 110 %. Inhibitors play a major role in high efficiency scenarios so you can check it by either increasing your template dilutions or try to use less amount of template.
Regards....
10ng/ul is my favourit template amount for most of the qPCR. The more template, the more inhibitors. Opitimize it.
Sincerely
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