What are factors increasing between-run or between-sample variation in
real-time PCR experiments and how can I minimize them?
I have only little variation in replicate measurements with cDNAs from
one tube but I have too high a variation in measurements with cDNAs
coming from different tubes but still from the same source. I had
several cell culture flasks with the same cells in them. I isolated
total RNA from each each cell culture flask separately and performed
reverse transcriptions for each RNA in separate tubes. But since the
RNA is from the same cell type, just from different flasks, I should
essentially get the same results. But the Ct (threshold cycle)
differences go up to 1.6, which considerably affects the results
because of the exponential nature of the formula used for evaluation
(2 to the power of minus delta-delta Ct).
May there have been signficant differences in the efficiencies of the
reverse transcriptions alhtough they were performed in parallel and
simultaneously? If yes, what can I do about this?