RT-PCR Problems with Reverse Transcription Step

Olivier Gandrillon Gandrillon at maccgmc.univ-lyon1.fr
Tue Sep 12 06:41:59 EST 2000


In article <B5DEC034.904%kendrick at u.washington.edu>, Wilson Clements 
<kendrick at u.washington.edu> wrote:

> I am having problems with reproducibility in generating cDNA from RNA.  I
> get variable amounts of cDNA from the same pool of isolated RNA.  I don't
> think the problem is in the PCR step, because wants I have made a pool of
> cDNA, I get very reproducible PCR.  Does anyone have suggestions for
> increasing the reproducibility of the RT step?
> 
> So far I have tried:
> 1) Increasing the dilution of the RNA to decrease pipetting error.
> 2) Using two different RTs (Superscript 2 and Stratascript).
> 3) Playing with concentration of Oligo dT 18 primer (200-600ug).
> 4) Including or not including DTT at 10mM final.
> 
> Any suggestions will be greatly appreciated.

You could try to make two consecutive round of RT, in case the amount of 
input RNA is not fully transcribed each time. In that case, just make a 
45 mn RT, then add some more RTase, and proceed for an other 45 mn

Hope this helps

Olivier Gandrillon
Gandrillon at maccgmc.univ-lyon1.fr






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