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primer selection for RT prior to real time - an insiders hint!!! (Oct/23/2006 )

hi everyone
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!!!)

cheers

-aussieuk-

QIAGEN already use this combo in their pre-made RT products.........

-John Buckels-