I have Young and Old mice samples analysed for same gene on two plates, could not fit them all. Is tehre any way to correct for the varitation and compare two plates?
Anne Hofmann yes I did but not on the same plate. I have 20 plates of 10 genes (incl ref genes), 10 for young and 10 for old mice. each plate has its own standard curve using cDNA. I am now calculting the ratio of difference in between curve points.Then, I would take that ratio as a correction factor to multiply with all other unknown points.
@Florian C Priller similar method to your suggestion but I divided the points rather than subtracting.
Hi Farya, in case you did pipette your samples very well without any variations, you can theoretically normalize your samples according to the housekeeping gene. Only take care, that it is a well chosen one which is not regulated with age (of your mice). If you are willing to optimize, use a multiple-PCR with primers for your gene and primers for the housekeeping gene in one pcr reaction (tube). Like this, you can exclude the pipetting variations from your data. For example, taqman PCR is used for this.
Hi Farya,
best would be to run at least one sample on both plates. This would allow you to refer samples on plate 2 to samples on plate 1, with the basic assumption that the same sample on the two plates has to be identical.
How many samples do you have. Try a 384 well plate. Hopefully that should take care of your problem. But you should have a reader for those plates
Jaya Aseervatham we dont have a machine which could analyse 384 plate. therefore, I set up on two 96well plates. Standard cuve(5) + 24 sample duplo(48)+ controls+ NRT on each plate. Plate 1 with young samples and Plate 2 with another set of 24duplo of older samples.