RT-PCR of Rare mRNA

Ng Hian Cheong medp3019 at leonis.nus.sg
Mon Feb 27 00:22:31 EST 1995


  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 
process.
  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.

Ng Hian Cheong
27Feb95



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