PCR product smaller than expected ?!?

CV Nadaraja cvnadaraja at HOTMAIL.COM
Tue Jun 17 22:57:22 EST 1997


Hello all:

I'm doing some RT-PCR from RNA isolated from a human prostatic CA  cell 
line, and for some reason, one of signals is less than half the 
published size.  I originally designed my primers with the Wisconsin 
Package software, and later with another popular program. Neither primer 
set works.  The size of the amplicon ought to be around 2.2 kb, but 
regardless of which set of primers I use, or how I manipulate the PCR, I 
get a very strong 500 bp product. What gives ? All the positive controls 
work fine, and I've even tried different ways of preparing the RNA and 
the cDNA. The RT reaction works best with random hexamers or a 3'UT 
gene-specific 15-mer -- well at least the controls and all other 
signals, even rare one work.  The gene of interest is a prostate 
specific membrane associated enzyme, whose signal we've detected in 
northerns using a conserved region in the rat as probe. Has anyone ever 
had this problem with PCR in general, and RT-PCR in particular?

Thanx in advance for any suggestions.

CV


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