What is the ideal amount of RNA to use for the RT? and how much cDNA to use then for the PCR?
I did RT with a solution of RNA of 0.36 ug/ul. Then for my PCR I used 1 ul of the cDNA obtained and used 25 cycles because I was hoping to see a difference between an endogenous level and an overexpression. However, I detected no band at the expected height of 500 bp but a diffuse band at 100 bp. What can this 100 bp represent? primer dimer? I switched to 35 cycles and I used either the same amount of cDNA or add 3 times more cDNA. In both cases I got bands at the expected height (but stronger when I put more cDNA) and still big bands at 100 bp.