I started to work with different samples and i look for the expression of different genes in these samples. Since I have new samples every week, I cannot accumulate them and do just 1 run for 1 gene on one plate for all my samples. Instead, whenever I have them, I run a housekeeping gene for each sample and I run my genes of interest.
So my question is, what are the rules for comparing the expression of a gene for samples done on different days (without changing a protocol or the machine)?
I have heard of inter-run calibrator but i am not sure I understood well what it was. I was told that the expression of this non-relevant sample on different plates tells you if your qPCR was ok and did not differ from other ones. If this is true the relative expression of this non-relevant sample will be the same between different runs and this will allow you to compare your true samples (assemble them on one graph).
So did i understand correctly what an inter-run calibrator is? What should this inter-run calibrator be? Can I take just a completely different sample (organ) and look for the expression of a completely different gene? or should the calibrator express the same genes that I am looking for?
(Someone in my lab told me that in order to be able to compare my different runs, I must re-run one of my samples with each new sample, but it is just impossible because my samples are small and if i do it i will compare maximum 3 samples. Is this person correct?)
Your answers will help me a lot
Thanks a lot
Edited by Liana, 02 May 2010 - 10:01 AM.