which method for relative quantitation? - (Nov/30/2006 )
Hello! Here the same problem. We use relative quantitation but often the choise of method is difficult. delta-delta Ct is good but not enough, with n-fold is not always possible to do variance analysis. We also try REST. How do you do?
Each method has its own pros and cons, and I don´t think there is a "perfect" method nowadays, it all depends in what your experimental design is like and what are your objectives.
I have used REST and standard curve method, and I personally favor the latter. I don´t like the way the REST method does the stats and normalises the data with the housekeeping. I really dislike the delta delta CT method, but I admit that in certain situations it can be very convenient and adequate.
Many thanks we'll try standard curve method with Y= ax+b. Then do you do the ratio between ng of target gene and ng of housekeeping gene? Have you some references about?
thank you very much
Check www.genequantification.com, you'll find fantastic information regarding the different methods to analyse qPCR data, including reviews.
I use relative quantification, therefore I do not calculate amount of cDNA (ng). I relate my unknown samples to an standard curve, obtaining a relative amount for my GOI and same for housekeeping genes. Then I do the ratio between GOI and geometrical mean of my housekeeping genes (I use more than one).
THANK YOU VERY MUCH. I know that site, but I never thought about standard curve!.... I'll try with it.