how much cDNA do you use for RT-PCR? - (Oct/01/2008 )
i'm using the taqman gene expression assay protocol for rt-pcr. a labmate of mine uses about 1500 ng of cDNA for each reaction while the protocol suggests 10-100 ng. i was wondering if using more cDNA will alter the Ct values. i would assume that the primer set + probe supplied with the taqman kit are the limiting reagent and not the amount of cDNA- thereby using less cDNA (as long as it is above the threshold of say 10-100 ng) wouldn't change the Ct values. problem is i need to run multiple pcr experiments and i dont want to have to keep going back to make more cDNA if i can get away with using less in the first place.
any suggestions or advice or comments???
thanks in advance!
the best thing to do would be to run a pilot experiment, titrating the amount of cDNA before you start using your actual samples. You want your cDNA at high enough concentrations so that they fall within the linear range of your system, but not so much that you are out of that range. I would suggest a 10 fold serieal dilution series. The primer and probe should be added in excess to avoid signal loss from their use, although you should validate your primer efficiencies as well.
Well it depends what you are looking. For example if looking for viral DNA or cDNA or you will need a bigger concentration than for a housekeeping gene. Always make an optimization run for primers, probes and templates. It will save you time and reagents.