primer selection for RT prior to real time - an insiders hint!!! (Oct/23/2006 )
went to a real time workshop last week with some pretty 'big' guys.
basically here is the inside info!
- accurate quantitation (obviously) depends on which type of primer you use in your rev transcription
so oligo dT if your amplicon is 3'; random priming if your amplicon is 5'; and gene specific if you have loads of 2ry structure etc etc
but....here is the clincher (and maybe most of you know this, but I sure didn't!)
the best all round primer to use (if you don't have time/money/enough sample to optimise oligodT vs random vs gene specific) is:
1uM oligodT and 10uM random nonamers
Apparently the nonamers are way more efficient than hexamers too by the way.......(even though all the companies are still selling hexamers!!!)
QIAGEN already use this combo in their pre-made RT products.........