non-linearity - Ct - (Nov/07/2008 )
[just learning RT-PCR]
if I have run a dilution series and I failed to observe linearity in the Ct values, how would I troubleshoot? What are the different factors contributing to this non-linearity?
Please.. someone could explain me that?
First look at the graph, how nonliear it is. (post a screenshot?)
1) More diluted samples exhibit higher efficiency (the curve is bended), otherwise its more or less linear - One of the reasons can be presence of PCR inhibitors. If you use cDNA use maximum 10% or reaction volume or cleanup the samples after RT. Use highly purified (measure 260/280 ratio) RNA to your RT reaction.
2) All of it it's more or less linear but with very high error rate - Common reasons can be pipetting error or incorect amounts of template in different dilutions. Check your pipettes, if they are calibrated right. Use low retention plastics and tips. Maybe try some other sample with the same pipettes and plastics, to see if it's still inconsistent.
Also a problem with sealing of the wells and subsequent evaporation during PCR can cause inconsistence. Make replicates of each of the dilution, and see how close to each other they are.
If you have very low concentration of the target gene, the results will always be more inconsistent. What are your Ct values?