Housekeeping gene always on the same plate as samples? - (Aug/01/2006 )
I am designing the first time a QPCR experiment, so I´ve two questions:
I want to analyse the expression of about 30 genes in 6 different treated grapevine tissues.
So I want to know: should the HKs always be on the same plate, in the same run, as the samples, or is it possible to creat one run with the HKs and use this results for the following runs (with the samples) in the delta delta method?
My second question is, what is the best cDNA concentration fpr QPCR?
Thank you for your answers!
I'm a firm believer that an HKG should always be run on the same plate, exposed to the same conditions, prep time, same Master Mix used to prepare the plate, etc. I also had this confirmed by ABI.
I use an input amount of 10 ng cDNA per reaction, but this should be optimized with your system, and also depends on the expression levels of your GOI's.