qPCR data analysis - (Oct/29/2010 )
I'm fairly new to qPCR and am having a hard time wrapping my head around the data analysis part. For the sake of simplicity, let's say I'm using 2 genes: 1 housekeeping and 1 gene of interest. First off, I open up my data in Bio-Rad iQ, switch the view to log, and analyze one gene at a time. How do I know where to set my threshold line to calculate my CT value? I know it is supposed to be in the linear part of the graph, but the graph is linear over a large range and changing the threshold in this range thus changes the CT values. Also, to compare my gene of interest to the housekeeping gene, should the threshold line be the same? If not, why not?
Once CT values are determined, I understand why you calculate the deltaCT of the target gene and housekeeping gene. What is the purpose of calculating the deltaDeltaCT, and what exactly does 2^-ddCT mean?
I'm also really confused about why you change the Y axis to log view. I know it has something to do with exponential growth is a straight line on a log scale, but can't figure of the math of why that is so. What are you taking the log of? I may be able to figure that one out on my own though.
Thanks so much for the help!
Look up the whitepaper: http://www.integromics.com/WhitePaper_qPCR_Statistics and the Livak paper. They are very useful.