RT-PCR of Rare mRNA

Rae Nishi nishir at ohsu.edu
Wed Mar 8 17:30:14 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 
> : 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.


We are also trying to do RT-PCR of a rare message.  Although optimizing
the PCR is very important, here's what we had to do in addition to
optimize the RT step:  use BRL's superscript (no RNase H activity) and
put in ALOT of total RNA (3-5ug).  If you put this amount of RNA in you
have to treat with RNase-free DNase to get rid of the last bit of
genomic DNA or use primers in two different exons so you can
distinguish genomic product from cDNA.  You could also prime the RT
reaction with a specific 3' primer in the sequence of interest (we
usually use oligo dT).

Rae Nishi
Dept. Cell Biology & Anatomy
Oregon Health Sciences University
Portland Oregon 97201
**that's Orygun, NOT Ora-Gone**



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