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Real-time qPCR data analysis - (Nov/16/2010 )

I am doing relative gene expression. I am currently in the process of determining what housekeeping genes are actually suitable in my study using the Vandesompele method. My questions are....

1) It has been suggested to find >3 HKG that are expressed at the same level across different experimental treatments and sample types. If I find 3 HKG that fulfill these criteria, will I have to run them each time I run my experimental samples?

2) I have been using the delta delta ct method to analyze my data. According to Bio-rad this method assumes that the GOI and HKG are amplified with efficiencies near 100% and within 5% of each other. What is "near 100%"...96-98%? Also, I have run two standard curves on my HKG to determine amplification efficiencies and some runs vary within 5% (92% and 95%). Should I re-run my standard curves to get a better idea of what my E really is? I keep everything standard between runs, but I always feel like there is going to be some variation in efficiencies due to pipetting, etc. What E value should I use in my data analysis using the delta delta ct? Do I have to run a std. curve each time I run my experimental samples? This would be a lot of reagents!

Thank you!



to your first question: you just have to run your HKGs just once but be sure that you always compare the same samples on one plate e.g. if you run samples 1-40 for your HKG on one plate you should not spread those samples on different plates for your GOI.

to the second question: in my experience, the standard curve reproducibility increases with the broader dilution range of your standard curve. I would accept efficiencies between 90 - 105%.