HKG validation - interplate normalisation (Nov/23/2009 )

Hi guys,

I want to chec if my candidate HKGs are stably expressed, so I have my control and treated samples in 3 biological replicates. i'm trying to use Genorm and NormFinder to screen the possible HKG between the samples. My problem is that I can run only 72 samples in each run: it means I can run only 3 candidate HKGs at time for all my samples (I have 6 HKGs). I'd like to know what I need to be able to compare the two runs. My confused understanding is that I need a std curve in each plate (with the same primers?) and the same sample in both plates (again, same primers?). and then do I fix the threshold so that it is the same for all genes or what?

I appreciate any hints,

Thank you

i guess u aer thiking on teh right lines.. ne more thing tat i would like to add is also make sure that the recovery of teh standard values in both the places are comparable. Only then the two plates can be compared accurately.
ideally a spike control of your sample is preferred but tehn you will end up by analysing only one sample per plate then.... so you can try to remove a conc or two from the standard and accomodating the spike control for each sample.
Also make sure that atleast there are 5 std points for a reliable curve fit!!!
Best luck!!

Thanks Pradeep Iyer,
I might be on the right path, but I still don't get what I'm supposed to do and how. I know I need the std curve to go from the Ct to the actual cDNA concentration, but I can only run a std curve for one gene. Can I infere the concentration of other genes from this curve?
And then, if I run a sample common to both plates shall I set the same thershold and keep record of the Ct difference for this samples in the two plates, and take it into account when I calculate the cDNA concentration of the genes in the second plate (using the std curve from the first plate or I need another std curve?)

If anybody knows where I could find anything more specific plesa, let me know.

Cheers