Problem with calculation using comparative method - When housekeeping gene is expressed less than sample (Feb/10/2006 )
This is my first post here. I was wondering if someone can help me out. I ran different samples of a particular gene: PARP with control cDNA and cDNA with a inhibitor to a pathway at different times to see how the PARP gene would be expressed differently if a certain pathway was inhibited. So for example i ran 3 samples of 24 hr DMSO or control of PARP rt-pcr and 3 samples of 24 hr UO or inhibited pathway of PARP rt-pcr. I ran these same exact samples of cDNA with another gene beta-actin, which is the housekeeping gene. My results show that the CT value for the beta actin is higher than the expression of parp in both the control and the inhibited cDNA sample (PARP CTs>ACTIN CTs). They also show that parp has a higher CT value if the pathway is inhibited in 24 hrs than the control (24hr UO CT>24 hr DMSO for PARP). How do i use the comparative method to calculate the amplication of the expression of PARP with inhibitor over no inhibitor when the housekeeping gene is expressed less?
Thanks for the help
it does not matter if the housekeeper is expressed more or less than your target; it matters only that it remains constant across your samples.
when you say 'comparative method' are you talking about delta delta Ct? try to plug the numbers into the equations, it should work for you
does this help you?