advice needed for check trace mRNA

James S. Sutcliffe jamess at mbcr.bcm.tmc.edu
Thu Apr 21 10:17:55 EST 1994


In article <leach-200494151630 at med-pharm5.bu.edu>, leach at mbcrr.harvard.edu (Martin Leach) writes:
|> In article <1994Apr18.151548.1 at leif>, ldeng at kean.ucs.mun.ca (GEORGE XIAOXI
|> OUYANG, ELECTRICAL ENGINEERING, PHONE 579-0970) wrote:
|> 
|> > Hi! Dear Netters! I want to identify very little trace of mRNA in the cells 
|> > by RT-PCR or Northern hybridization. I certainly want to show the RT-PCR 
|> > signal is not come from DNA. So anyone can tell me what is the best way to |> > extract RNA which is free of DNA?
|> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
|> 
|> Virtually impossible!
|> 
|> 
|>  What is the best probe (for Lymphocytes) 
|> > for the very-low-expression mRNA in Northern hybridization? Thanks in 
|> > advance!  Langtuo Deng at: ldeng at kean.ucs.mun.ca
|> 
|> 1. Either try RNAse protection - more sensitive than Northerns.
|> 
|> 2. Or try RT-pcr with primers that span at least one intron. - so if DNA
|> contamination it either will be larger product or Un-PCRable if too big.
|> 
|> Martin Leach
|> 
|> -- 
|> 
|> .....          Martin Leach                Email:leach at mbcrr.harvard.edu 
|>    _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
|>    / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
|>  _/  |-/__==/  80 E. Concord St. (L603)
|> (BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
|>                USA                           head.....WIBBLE" -BLACKADDER  


It isn't impossible, however to remove contaminating genomic DNA from RNA
preps.  The trick is to incubate your RNA with RNase-free
DNase, before you synthesize first strand cDNA. The Promega (no
affiliation!) RNase-free DNase works well for this purpose. I do this
routinely for RT-PCR, especially when I have no knowledge of intron/exon
structure. Would be glad to provide more details if you're interested.

Hope this helps.

Best,

Jim Sutcliffe

**********************************************
* James S. Sutcliffe, Ph.D.                  *
* Howard Hughes Medical Institute            *
* Department of Molecular and Human Genetics *
* Baylor College of Medicine                 *
* Houston, TX 77030                          *
*                                            *
* Tel: 713-798-4396/4795                     * 
* Fax: 713-797-6718                          *
* internet: jamess at bcm.tmc.edu               *
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