RT-PCR of Rare mRNA
sullivan at gwis2.circ.gwu.edu
Sun Mar 5 17:26:22 EST 1995
Ng Hian Cheong (medp3019 at leonis.nus.sg) wrote:
: I have spend few months trying to RT-PCR a mRNA which if present may be
: at a extremely low levels. So far, the results have been discouraging.
: The method I have used is Nested 3'-RACE ; i.e using oligo-dT and two
: gene-specific primers designed from my genomic clone in 2 rounds of PCR.
: Using two gene specific primers only for alpha tubulin,I was able
: to obtain the cDNA of alpha tubulin showing that my Reverse Transcrpition
: was alright.
: Currently I am not sure whether my failure to RT-PCR this mRNA was due
: to absence of the mRNA or due to failure in optimization of my RT_PCR
: Can anyone tell me how to optimize the individual steps involved in RT-PCR
: process to allow me to obtain cDNA of rare message ?? Any good control
: for RT-PCR of rare mRNA ? Any suggestions will be MUCH appreciated.
: Many Thanks.
One control would be to add progressively smaller amounts of a known RNA
(such as a-tubulin) to your samples and try to RT-PCR up a fragment from
it using the methods you normally use. THis will give you a ballpark idea
of the limit of your technique (although there will be differences that
depend on the precise sequence you're amplifying).
If you've already acertained that your input RNA is intact (which you
should do via formaldehyde gel electrophoresis), then it's likely that
most of your optimization will be at the PCR steps.
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