How to test whether total RNA is changed or not? - (Apr/16/2007 )
I treated cells with 5-aza and then extracted RNA, then cDNA synthesis and real-time analyzed gene expressions. I tested 2 house keeping genes in the real time: HMBS and GAPDH. Both genes were down-regulated after the 5-aza treatment and this happened in 3 cell lines I've tried. I feel suspicious that the 5-aza de-methylation turned on expression of many usually non-expressed genes and made the total RNA per cell increased. Then the increased total RNA made me biased use less RNA to do cDNA systhesis and real-time analysis. That made hkg that actually unchanged seems to be down-regulated in the real-time. So how can I test whether 5-aza affected total RNA per cell or not? (Hope this makes sense!)
ANY suggestion, please, please, please!
you could measure the amount of RNA obtained and normalize by the number of cells if you know both...
Do you need to do absolute quantification? Then your suspicion makes sense in my eyes. But this would be no problem if you are going for relative quantification of your genes of interest.
Yes I think you are right krümelmonster! No need to do anything about it, which is good!