RT-PCR

gordon allison 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...
>
>Dear All,
>
>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




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