RT-qPCR is a gold standard for measuring mRNA abundance of a targeted gene, and it has largely replaced nuclear run-on assays. While there may be some overlap among the two assays, a nuclear run-on actually detects frequency of transcription initiation, while RT-qPCR does not. If I want to detect real-time gene expression in a whole organism and I want to avoid using radioactivity (e.g., nuclear run-on assays) what are my options?
Hi Hussain,
Thanks for the advice. Instead of Westerns I do antibody staining of my genes of interest, but that is only semi-quantitative. I'm seeing a lot of up and down regulation in these genes over short time intervals with overall increases in protein levels. so I need to make sense of that. The problem with inferring gene expression from RT-qPCR alone is that it doesn't take into account RNA decay. Another option is that I add a RNA polymerase inhibitor prior to RNA extraction, run RT-qPCR, compare it to non-inhibited samples and subtract the difference.
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