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mRNA for Differential Display

Douglas Rhoads drhoads at MERCURY.UARK.EDU
Tue Jul 23 07:16:09 EST 1996


> Hello yeast newsgroup
> I have been looking at protocols for RNA extraction suitable for
> S.cervisiae which will give me good undegraded mRNA for Differential
> Display. Most do not mention mRNA quality. I am not experienced with
> yeast but I think the method needs to be fairly harsh to break down
> the cell wall but I am concerned that the mRNA will be damaged by
> shearing. My supervisor has suggested an enzyme treatment to soften
> the cell wall followed by a detergent type of extraction.  If you
> have any experience or thoughts on this problem I would be most
> grateful to hear about them. Cheers, Dianne  
> 
If you really intend to do ddRT-PCR then you need very good quality 
RNA and since the technique usually requires differential treatment 
of two populations then you don't have hours to treat the cells and 
loosen the cell wall with enzymes since the cells will be 
reprogramming their gene expression during the 30-60 minute enzyme 
incubation in Zymolyase or Glucuronidase.  

We have used acidified phenol and freeze thaw to break the cells, a 
technique described by several investigators and I believe is in the 
Yeast edition  of Methods in Enzymology.  I don't have the reference 
here handy.  You might also be able to get away with a glass bead 
vortexing to break open the cells.  Regardless, it is better to give 
up yield for quality in size.  We usually then desalt and remove 
small oligos by running over a Size exclusion spin column to get rid 
of small fragments (<200).




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