Reference Gene - How close is close? - (Apr/04/2006 )
Hi all. I have been looking at Actin, ATPase, and GAPDH as a housekeeping gene for tomato leaf tissue.
There is a difference between the control sample and the test sample in all of genes of 2 to 3 Ct values:
Actin (50nM) control sample Ct is 30.1 test sample Ct is 27.1
ATPase (200nM) control sample Ct is 27.1 test sample Ct is 25.2
GAPDH (300nM) control sample Ct is 26.8 test sample Ct is 23.3
note : cDNA is 100 ng/u
Is this an acceptable variation? How close is close? Do I spend the rest of the year screening for housekeeping genes?
Thanks for any feedback!
First thing is have you only performed n=1 for these? If so then you really need to do a minimum of n=3 before you can start to decide on trends.
Thanks, Doc. The values represent one test run in triplicate. Do you mean that I should repeat the test on three separate runs?
I'd say that depends on the variability of your results (so how close your seperate results are to your mean), but don't think its nessecary. And your variability looks allright, but it depends on the effect you are investigating. To be sure you can normalize for all three genes and for every gene seperately, to determine the impact of your housekeeping variation.
Hi !!! For most of experiments with relative quantitation Ct diference between control and experiment for hosekeeping genes should not be greater than 0,5 Ct, although diference of 1 Ct is also acceptable. Try to use RPLP0 as your reference. I had many problems with GADPH (very variable results) and even more with ATPase.