RT-PCR protocol

January Weiner nospam_jweiner1 at ix.urz.uni-heidelberg.de
Fri Feb 1 12:33:33 EST 2002


Hi,

I'm trying an RT-PCR of a bacterial gene with random hexamere primers
(Roche) and MMulv, 2-tube from 1ug total RNA.  The reactions seem to work,
but (there's always a `but'):

	- the reactions seem not to be reproducible
	- ...and not very efficient.

I'm struggling to quantify the cDNA using real time PCR.  The PCR reactions
seem to work nicely (standard curve is OK), and I get very similar results
using different primer-pairs (different reactions) for the same cDNA and
the same gene.  "No template controls" (NTCs) seem to be clean (no
contamination).  However, the values are very, very low, and I got strange
artifacts.  For example, even though the measured cDNA quantity for the two
different primer-pairs is the same, it is lower then the measured quantity
in the NPC (no polimerase control), which, basically, is impossible or very
unlikely (in 18 different probes???).  

I arrived at the conclusion that the reason for my troubles is an
inefficiently designed RT reaction, because my real time PCR measurements
seem to be highly reproducible with small or no errors.  Therefore, my plea
-- I'm looking for a good protocol for a cDNA synthesis of bacterial genes
using random hexamere primers and, preferably, MMulv.  

Thanks in advance,
j.

-- 
----)-\//-///-----------------------------------January-Weiner-3-------
His ideas of first-aid stopped short of squirting soda water.




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