I'd like to analyse my data using the 2-ΔΔCT method, but I'm a little confused as to the Ct value I should enter into my excel spreadsheet when my samples do not amplify.
I'm working with the following cell types/conditions, looking at the gene expression of lots of other CD molecules :
[control] 1) CD8+ CD56- cells
[case] 2) CD8+ CD56+ cells
[case] 3) CD8+ CD56- cells which have been chemically stimulated
[case] 4) CD8+ CD56+ cells which have been chemically stimulated
Ideally, I would like to compare 2, 3 and 4 to my control sample, number 1. My HK gene amplifies perfectly in all cell types/conditions, each producing consistent & reliable Ct values. My problem is with my GOI - depending on which gene I am looking at I can see up/downregulation or no expression at all. No expression is quite common in samples 1 & 2, which then means I have no Ct value to enter into my spreadsheet (which I of course need, so I can compare all my case samples). What do people do in this situation? Would you enter a Ct value of 0, or enter the value of the total number of cycles you have run? Or should I change my control to another sample, say 3 or 4, which regularly amplify?
Any help and suggestions would be greatly appreciated. I'm getting some weird results (eg, 159146-fold upregulation - I'd expect upregulation, but this much is insane!) which I think cannot be right.
Thanks in advance,