validation of micrarray data by realtime pcr - (Feb/03/2007 )
i have done microarray experiments to identify genes that are differentially expressed in response to a pathogen and now i need to validate this data by real time pcr.
the question is do i have to do serial dilutions for each gene for every rna sample i have got ( for me its 5 time points after treatment or mock treatment) and compare it with standard curve of the house keeping gene. if so i need to do 50 reactions per gene. and do i have to repeat the whole set of rt pcrs a second time or do i just do it only once.
or do i just serial dilution in only one rna sample , compare its with house keeping genes and using that as a standard to calibrate in all the rna samples.
You only have to do serial dilutions of one sample for each gene - for example i have just done a similar experiment where i had some spare cDNA (from the same tissue type as my samples), i made a serial dilution from this and ran each gene with this sample to generate the std curve. i also used this sample to create my reference gene std curves, and i will use these results to calibrate all the RNA samples on my final plate.