I've been working on IL-13 gene expression analysis using SYBR green master mix and step one plus device, I tried 3 different sets of primers but still there is variation in cycle threshold between technical replicas although the housekeeping gene hadn't.
sometimes the cycle threshold is undetermined. I replicated my plate three times but still.
Any help or recommendations?
if it is regarding the nature or molecular biology of the primer or gene itself I would be happy to know :)
Article On non-detects in qPCR data
I think this article will be helpful.
I would try with different primers and definitely have a look at the dilution of cDNAs. And of course, the variation in technical replicates might be due to the pipetting errors.
To validate the primer, you can run the normal PCR and gel.
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