RT-PCR troubleshooting

Wangxue Chen w.chen at clear.net.nz
Wed Jun 2 17:59:21 EST 1999

I am not an expert in RT-PCR.  In our hands, one-step RT-PCR appears to
require more precise conditions than traditional RT and PCR, which are
more robust.  My suggestion is that you may want to try it using
traditional two step RT-PCR first to ensure you get the mRNA in your
sample and the optimal condition.  The two-step will alllow you more
easily to dissect where the problem is (i.e. in RT or in PCR).

Good luck.


In article <tburmei-0206991759240001 at tburmei.dialup.fu-berlin.de>,
tburmei at zedat.fu-berlin.de (Thomas Burmeister) wrote:

> Hallo !
> What kind of primer do you use for the RT reaction (oligo-dT, random
> multimers, specific primer, ...)?
> If you want to detect a specific mRNA, you will get the most efficient
> reverse transcription with a specific primer. You should use the same (3')
> primer, that you are using in the subsequent PCR.
> Thomas Burmeister
> In article <37545FBF.9BBD2C24 at pharmacy.uq.edu.au>,
> s.roberts-thomson at PHARMACY.UQ.EDU.AU (Sarah Roberts-Thomson) wrote:
> >I am trying to amplify a particular mRNA target and having little
> >success, although other targets cause us no problems. I know the mRNA is
> >present in the sample RNA we are using. We are using a one step RT-PCR
> >kit and can show that the PCR step is working with our primers by using
> >a brain cDNA library. We also know that all the components in this kit
> >are okay as we can use it to amplify other targets. Does anyone have any
> >suggestions? We have tried a range of magnesium conc, annealing temp etc
> >etc.

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