I am trying to develop a standard curve for miRNA using qPCR. I keep getting Ct vs log(mass) slopes around -5, which is much lower than -3.3 (100% PCR efficiency). I am looking for advice on how to troubleshoot this.
I think it does. Some time ago, ABI even produced qPCR machines that worked somehow that way: they always worked with an annealing temp of 60 °C and adjusted the concentrations of the primers. At least that's what I remember. So you could give it a try. But there are many more. MgCl2 might play a role, or you could add BSA, which also helps.
And of course: how well designed are your primers? I would start with a large set of primers and look for the best ones. I used primer3 and tried to obtain a couple of primer pairs with high scores. Ideally, one forward primer even works with two or more reverse primers and the other way around. Like this, you can try many combinations and look for the best one.
Primer concentration does not usually change the efficincy of a pcr because the primer is always in huge excess so small differences in concentration do not usually make much difference. Having said that if you have far too low an amount there will be little amplification and if the primers are badly designed then having too much primer will form primer dimers which amplify very well so remove huge amounts of primer very quickly and will also lead to low efficiency amplifications