normalization between 4 different run - (Dec/13/2006 )
I got 10 samples per treatment group for 4 groups Therefore I have to load these samples in different plates (different run). I am using standerd curve for each run and HPRT as house keeping gene. But for anaysis, I have to normalize the result, for the result come from different run. anybody got any idea how to normalize the data between different run, using another reference sample? Is there any useful calculate Exel sheet like REST for that normalization? Iam looking for this normalizarion desperately!!!Many thanks.
well assuming you're doing RTqPCR i was wondering if you make your own ref between plates by considering 4wells more per plate.
In those 4 wells you take 1sample per group and thus can compare these 4 wells in the different plates.
i've attached a picture in order to try a better explanation but we can discuss more if you want
In those 4 wells you take 1sample per group and thus can compare these 4 wells in the different plates.
i've attached a picture in order to try a better explanation but we can discuss more if you want
Thank you very much! I really appreciated your suggestion. I think I understand your picture; want a further discuss with you.
I actully want to run standard curve(GOI and House Keeping gene) with the same sample on each plate to compare the exfficiency of each run. and load one sample as reference sample on each plate. Then the samples in the same group have to be seperated in different run. I mean, the data for samples in the same group come from four different run , which has to be normalized to be analyzed together. I got some idea to use comparative quantification method(delta-delta-CT) which assume efficiency is 2, just set up an reference sample in each plate, then all the samples are divided by this reference sample. But for my samples, the slope of standard curves between GOI and HK gene is bigger than 0.1, therefore I can not use delta delta CT to analyze. To use standard curve method, do you get soem idea how to analyze the data? many thanks. THE ATTACHED IS ONE OF MY PLATE.(USING abi 7500)
well seems little complicated but her is what i think
you have to compare the control reaction reproducibility using a standard derivation measurement
then if results are reproducible you can take the mean of each well over the (also check the variance and then proceed as if it was one single plate.