PCR replacement for run-ons???

John Fagan jfagan at miu.edu
Thu May 12 15:05:06 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 5Õ end of the nascent transcript of interest to ÒcountÓ
transcription complexes?  It seems that this would have many
advantages, including improved sensitivity, lower background, etc. 



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