goa at aber.ac.uk
Mon Feb 26 09:07:37 EST 1996
In article <Pine.SUN.3.91.960223132832.28564C-100000 at dsets.ulst.ac.uk>,
edq424 at dsets.ulster.ac.uk says...
>I have been increasingly facing problems with RT-PCR. It worked after
>three trials at the very begining. Now, I am trying it for more than 4
>times with no success. I have noticed that if I change the quantity of
>the first strand cDNA into the PCR reaction that the intensity of the
>band changes. Amazingly, the less the quatity of the cDNA the stronger
>the band up to a certain threshold.
>Is the first strand cDNA quantity so crucial in the PCR reaction or am I
>doing something wrong? How do you know that the result of PCR is optimal
>and really representative of mRNA monitored and is not due to imperfect
>PCR reaction? Is using positive control for a gene which is switched on
>all the time enough for quatitification of RNA?
>I am really lost in a PCR which does not work and the bias in results....?
>Give some details of your protocol and I may be able to suggest something.
rt-pcr can be very difficult to get going, it certainly was for us; now we
can't understand what we were doing wrong as it works nearly every time.
However, I well remember months of swinging from dazzling success to dismal
failure with the same batches of RNA from one day to another.
Good luck, Gordon
More information about the Methods