Hi Adrian,

If you are working on RT PCR, I suggest length should not go above 200 to max 250 for better result. Concentration of your primer should be 5 uM ideally for RT PCR Experiments but you can very the same to 3 uM to 7 uM working stock, dNTPs ideally 10 mM working stock.  Try to adjust fine tuning between the same..    

What about your RNA? what quality/quantity methods did you use? do you have reference genes?

You need to standardize the amount of RNA that you use in preparing the cDNA. Afterwards, you test the cDNA quality by using housekeeping gene primers. For the primers concentration and master mix try to use as recommended by the kit.

Hi Maulik. Thank you for your answer. What about MgCl2? should I use it in excess??Carol and Azzreena, thank you too for your answers. The housekeeping genes I'm using are GAPDH and B-2-microglobulin, and the cDNA is synthesized using RNA 2uM. The RNA quality is verified with an agarose electrophoresis and by 260/280 ratio.

Hi

I agree with Maulik, You should not go beyond 200 PCR product. ( Ideal range is 70-150, optimum 100). Now primer concentration, generally when I start working with new target I run a TEST run with 3 different concentration (200nm, 100nm,50nm). I have seen people use up to 5 uM of primer but if you go with higher concentration you may end with with Primer-primer dimer and that could mess up your Ct Values. As far as the concentration of other things, you can look up any kit available( I use Invitrogen) and follow their protocol and concentration you will be fine.