Looking at the enrichment of mRNA that includes a target alternative exon using qPCR, I see >100 fold increase of inclusion of the exon in an experimental cell-line. However, looking using reverse transcriptase PCR to determine the percentage of inclusion of that same exon I see a percentage difference between inclusion and exclusion of about 10%. How can that be?
Revers transcriptase PCR is semi quantitative because it differs with the number of PCR cycles you use (could be already saturated) and also relies on efficiency of the run and EtBr steining etc. qRT-PCR is quantitative since it directly measures the threshold cycles. Read more on the methods will help you understand the disparity. Wikipedia has a good entry on that
https://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction. However from both methods it seems that there is a differential expression. I would repeat the qRT-PCR and see whether the result holds. Then I'll rely on that.
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