I successfully performed RT-PCR on different cell lines several times, but now it is not working anymore. Some cDNA are amplified while others are not.
So, first I changed the enzymes (either for RT or for PCR) and the respective buffers, I repeated the RT-PCR and I got the same result again: only some cDNA were randomly amplified.
So, that means that the PCR is fine and the problem is only on RNA preparation.
I usually use:
TRIZOL
Chloroform
Isopropanol
75% Ethanol (made with DEPC water)
RT-PCR water to resuspend the pellet
Can the DEPC water used for 75% Ethanol be contaminated with some RNAase?