Random primers-real time PCR - (Apr/28/2005 )
When we use random primers then we will get different sizes of cDNA. What happens if the random primers will produce more small and useless sizes than necessary? Do we have to use this cDNA or do we have to exclude these small sizes cDNA by some kits before running real time PCR?
if you are talking random hexamer primers, then you lower your primer concentration or increase the amount of RNA used in the RT reaction. This will produce longer cDNA fragments as this lessens the chance of any random primer binding at any particular binding site. Thus giving cDNA synthesis upstream a better chance to synthesize a longer fragment.