Reference genes and dilutions in qPCR - (Nov/05/2010 )
Hello! I have been using beta-actin for my reference gene and would like to know: a) which reference genes others use, and, b) at what dilutions. I dilute my cDNA for the beta-actin 1:2000 and 1:4000 due to the gene's ubiquity and prevalence. However, I'm not getting consistent Ct results -- some are okay whereas others are not. I presume this is because the cDNA is highly diluted. Has anyone here experienced this? Any suggestions as to what can be done about the dilutions other than to simply not dilute it as much? Any way to make sure the cDNA is evenly diluted/diffused in the water?!
Any help is much appreciated.
There are commercial panels available on the market with so-called "housekeeping genes". It has been shown that often they are not suitable for any type of tissue or condition and need to be validated every time.
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