Help with delta delta ct analysis - (May/23/2011 )
I am trying to do a delta delta ct analysis on my data. However some of my control samples have very low expression and some of them don't amplify at all. This is good, becuase I'm trying to demonstrate higher expression in my treated samples, so this is matches my expectations. However with relative analysis how can I analyse the difference between two samples if the expression in one is zero? Additionally some of the control samples and and some of the treated have very high ct values. I run the pcr for 40 cycles and some of the samples only amplify after 30-33 cycles. However in this region the amplifcation is highly variable with the replicates having a very poor presicion and some of the replicates don't amplify at all. I'm not sure how I analyse the samples which have very late ct values?
Any advice who be greatly appreciated.
On the recent qPCR seminar a statistician lector from tataa biocenter mentioned, that for missing data they assume Ct as final number of cycles + 1. So if you have 40 cycles, the Ct of the missing ones would be 41.
(I found a forum post saying something similar)