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DDRT-PCR

Joel Guss jg183 at columbia.edu
Wed Dec 3 21:32:04 EST 1997


Thanks for the information on your success with the technique.
I just want to make sure I have it clear.  Most publications I
have read use the 12 anchored primers along with about 30
arbitrary 10-mers.  So, you do, overall, 360 PCR reactions times
two different conditions equals about 700 PCR's.
And if you want to avoid false positives, it would make sense
that you run, side by side, at least 2 separate RT-PCR sets,
which at least doubles this number.  Is this what you do?
You are right that it is a rewarding technique, but it is a
lot of work.  And one of the main strong points of the technique
that are pointed out are that you need very little RNA to start
with, but then what if you have 100 bands that appear differentially
expressed on the gel due to the high rate of false positives?
There is no way that if you are limited in the amount of RNA you 
have that you will be able to verify all of these on a Northern.
Also, one more time, how many 10-mers did you use and exactly
how did you go about selecting them (totally randomly or was
there some method)?
Again, thanks a lot for the reply and good luck with your work.
			-Joel



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