I have a problem with my qPCR. I checked expression of GOI by qPCR and it seemed that it is induced. When I tried to repeat experiment (with new set of RNA, but from the same cells' population) the result was quite opposite - there was silencing. When I run NB it was visible that silencing is what really happens (both sets of RNA samples). I have now wonders - what did I wrong in the first experiment so I get induction? I used the same primers (and I checked their efficiency), the same machine, the same reagents, RNA was not degraded. The only diference is that I prepared two stocks of RNA in Trizol, in first experiment I used the first one (and then I got induction), after that I thawed second sample, isolated new RNA and I used the second one (and I got silencing). For me it looks like I'm doing sth wrong during RNA isolation, but what can it be? Guy from my lab told me that prolonged exposure to Trizol can make RNA breaks so it may mix results. What do you think ?
I would greatly appreciate any help!