I'm having a problem quantifying mature miRNAs using stem-loop RT-PCR primers specific to several miRNA (I multiplex several stem-loop RT primers in one reaction), followed by SYBR GREEN qPCR analysis. For some of the miRNA candidates I get nice specificity (checked by cloning and sequencing), but for others, apparent from the sequencing, it seems like the forward amplification primer anneals to different places on the stem-loop RT primer. I've tried 'normal' specificity things; 1) higher annealing temperatures (up to 70 deg/celcius) which helps for one candidate, 2) lowering stem-loop RT primer concentrations in the RT reaction, thinking that the massive overload of RT primer could be the basis for the chance annealing of fw primer to the excess RT primer, but this only affected the CP values (higher CP values), even for the candidates with nice specificity.
So, my question is: What to do?! How can I increase the specificity? Should I try changing the ion concentrations in the qPCR reactions? Use Locked Nucleic Acids (LNAs)? Anybody got experience?
Thanks a lot