Hello all,

I am trying to design qPCR assays for a family of inducible innate immune peptides which are expressed at very low levels basally. When running standard curves to calculate primer efficiency, I’m finding that 10-fold and even 5-fold serial dilutions don’t yield enough points with detectable expression and tight replicates to generate a reliable curve.

So far I have been using cDNA from RNA extracted in the same manner I will use for my experimental material to ensure a comparable matrix. Given the situation, would it better to use cleaned-up PCR product?

I normally dilute my cDNA 1:10 and have added only one point with more than usual cDNA/less diluted cDNA as when I go above this I see inhibition. Would a narrower series of dilutions be reliable in estimating efficiency? I’m obviously hoping that one or more of my experimental treatments would drive expression, in which case I could go back and run a broader dilution series with the appropriate cDNA.

If anyone has been in a similar situation previously their advice would be greatly appreciated.

Many thanks.

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