18S as hk, how can i optimise it? - (Sep/18/2006 )
Hi all! i just made a qPCR, in which mi target gene was nicely amplified (BDNF), but the 18S curves (see attached file) may not be good enough for the quantitation......(although ive been told in my lab that i could use them,..... but want ur expert opinion)
I see many peaks, however in many samples there is a clear single peak, as in the standard. The negative control curve is the red one at the botton. The rest corresponds to samples and the standard.
I got primers for beta actin that worked very nice in the PCR, but for qPCR they didnt. Justo get no amplification at all (weird).
GAPDH primers dont work good on the normal PCR, and ive read that 18S seems to be one of the most reliable housekeeping genes (im comparing normal and regenerating after transection retinas/optic tecta). Any idea/sugestion on how can i improve my curves?? those peaks doesnt seem to correspond to dimers...what can it be? in a gel, i see very strong single bands with a little, little bit or smear behind it (may be due to the abundance of 18S?). The primers were new so i think thats not the problem...
Thanks for your help!!!
.......ops. I´ve been reading that using oligo-dTs in the RT step avoids rRNA to be amplified -cause has no poliA tails in it-. Then, it seems that i cant use 18S as a reference gene, is that ok??? maybe that´s why my curve look kind of weird........