I want to do a qPCR to detect expression levels for a gene transcript in zebrafish. I have wild types and homozygous mutants for this mutation and it is a nonsense mutation. Since the mRNA transcript will still be synthesised regardless of the mutation, is qPCR a good strategy to proceed with or am I better to apply the procedure to a downstream target of the mutated gene? Thank you in advance.
I guess why not. The relative expression of your wild type versus mutant mRNA of your samples by qPCR will reveal it clearly as you can observe an unequal expression between them based on your wild type control..And also by comparing the size differences of PCR products of both wild type and mutation samples on gel it will be confirmed anyhow. Usually it is stated that unequal abundance at protein level between the wild type and mutant alleles is characteristic for mutation.....
qPCR is absolutely relevant for such experiment. However, it depends on the type of mutation. If it is just a one-point mutation, it could be advantageous to design functional qPCR primers (in such cases hydrolysis probes are recommended, but based on my experiences, the dye-based detection could be functional as well). If there are larger changes in sequence, everything is much more easier;)
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