Why is the relative expression in qPCR so low?

Question

When analyzing different mutations through qPCR I found the $2^{-DeltaDelta ct}$ value for each mutation. All the mutation have similar values compared to the wild type gene, however one of the mutation has an extremely $2^{-DeltaDelta ct}$ value. Having $1$ for the wilde type, and an average of $0.9$ for some mutant genes, the exceptional case has a $0.05$ value.

I think one could argue that this mutation deletes a sequence from the wild type gene, but I believe other things can be suggested from this.

mutations pcr

I think one could argue that this mutation deletes a sequence from the wild type gene, but I believe other things can be suggested from this.

Krzysztof Czarnecki · Answer

It may be a particular mutation that affects expression altogether. Through qPCR you're able to tell the relative difference in mRNA levels of a particular gene between, say, a WT and a mutant. It may be that this particular mutation damages some regulatory sequence within the body of a gene (e.g. a downstream promoter element) or some regulatory sequence upstream. To me, this suggests that you're dealing with such a case here. Anyways, you'd need more data to tell for sure. You should have the mutation mapped, if you haven't already and check if the primers you used were okay. This mutation clearly hampers expression, as long as everything was okay with the procedure.

Answered by Krzysztof Czarnecki on December 16, 2020

Why is the relative expression in qPCR so low?

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