PCR-based replacement for run-ons???

John Fagan jfagan at miu.edu
Thu May 12 14:17:25 EST 1994


We routinely use the run-on assay to measure transcription, but find that
the assay is finicky and laborious, and that it subjects the experimenter
to high levels of 32P.  The essential principal in this assay is that you
are labeling nascent transcripts by extending them with 32P-labeled
nucleotides.  The extent of incorporation is thus approximately
proportional to the number of transcription complexes actively
transcribing the gene of interest.  My question is whether anyone has
tried using quantitative RT-PCR, using intronic primers from near the 5!
end of the nascent transcript of interest to !count! transcription
complexes?  It seems that this would have many advantages, including
improved sensitivity, lower background, etc.



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