Im starting a new project with all new samples and genes and starting up a qPCR for validation purposes.
When synthesizing cDNA cDNA I used 600ng (equal amounts of RNA).
When plating the qPCR i used 2ul of cDNA for a total volume of 20ul per well.
It was a taqman run for 5 genes (two testing genes and 3 housekeeping).
Problem is none of the genes worked in any of the samples. All CT's were above 40 !!
I will now do it wright and start by doing a standard curve with different cDNA dilutions but i just dont know where too start.
Is my problem caused by very high or very low cDNA concentrations ??
Should i start with for example 9ul of cDNA (higher i can go) and down to like 1:2000 or something like that?
Or should I just go from 1:20 to 1:2000 diluted cDNA for example.
Help please !!