RT-PCR with random hexamers
xxxgifalone at tin.it
Thu Jun 19 13:16:48 EST 2003
"Wolfgang Schechinger" <hubahopp at gmx.de> ha scritto nel messaggio
news:220.127.116.11.20030618232354.00a59200 at pop.gmx.net...
With specific primers, you have the chance to directly amplify your gene of
interest whereas with random hexamers, you'll amplify all mRNAs at random
positions. Then your signal/noise ratio is worse than with specific primers.
Sometimes hexamers simply don't work. I observed it several times when
doing specific and random priming parallel in RT. Of course, that's not a
good explanation. If you really need to use random hexamers, this might
help: add them, do a short heat step to melt secondary structures, ice
shock immediately, then perform the RT.
If possible, you could try random pentamers + a fixed base. (A,G,C or T) so
select just a subset (maybe there's a differential display kit sitting in
the back of your freezer where some of these odd primers are left over) or
even hepta or octamers, if Tm might be the reason for not working.
I haven't got any band neither with 4,5ug of total RNA.
Tomorrow I'll try random hexamers with 18S Internal Standard RNA and primer
pair so I'll see if my random are good.
I'll also try to increase RT amount (with my gene specific primer I get good
retrotranscription levels even with 0,2U/uL) but maybe with random I need
1U/uL for successful RT step.
I'll report results. Thanx.
xxxgifalone at tin.it (remove xxx)
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