Cell count vs Reference gene - Cell count vs Reference gene (Apr/16/2007 )
I wish to use Real-time PCR to quantify cytokine mRNA from purified lymphcytes. If I have accurate cell counts is it necessary to use a rference gene for absolute quantification amongst the different experimental groups?
I would say yes because if you rely only on the same cell number you don't have information about RNA extraction efficiency. after extraction you should quantify your RNA yield but this is often not very accurate, especially if assessed by spectrophotometry.
Also the efficiencies during the reverse transcription step can vary considerably, depending e.g. on RNA quality, contaminants,.... nevertheless, i would try to start with the same cell numbers.