I have done microarray to check differential expression in a given condition. I got some genes have changed differentially upto 10 folds. However, when I checked it by qRT-PCR, I found there is no remarkable change in their expression. What are the causes behind it? What else I can do for further confirmation? Is there any possibility of DNA contamination in my RNA because I haven't use DNase?
I know from Affymetrix gene chips like Human genome U133 A2.0 chip which I used during my thesis that more than 22.000 probe sets encoding for approx. 14.500 genes are represented and with the U133 A2.0 plus chip even more than double amount of probe sets encoding for approx. 22.000 genes are on the chip. So, several genes are represented more than once, with different transcript variants. And depending on the specific probe of the chip I could really see that sometimes on one probe the gene was differentially expressed and on the next probe it was not.
And therefore, a simple question is - when you were designing primes, did you design them for specific transcript variants or might this be a possible explanation?
Perhaps this link helps:
http://www3.appliedbiosystems.com/WebTroubleshooting/NTCPositiveAmplification/Determining_genomicDNA_contamination_RT_controls.htm
Thanks Vincent Block, Your suggestion has helped me to rethink about designing of my experiment.
While done the qRT-PCR, I generally treated the total RNA with DNase.
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