Stationary Phase Cells and mRNA isolation

Bower, Patricia Bower.Patricia at MBCO.COM
Thu Jul 17 14:17:37 EST 2003


We are doing some microarray studies on our production fermentations.  My
main problem is isolating mRNA from the yeast (industrial strain) during the
latter stages of the fermentation; at least isolating it consistently.
These cells are in stationary phase.  At this time we are doing a hot phenol
extraction followed by a chloroform and an ethanol precipitation.  The RNA
is washed with 70% ethanol and resuspended in DEPC-water or 10mMTris pH8.
The mRNA is isolated using Qiagen Oligotex columns.  Though RNA is present
by a A260 reading part of this is rRNA  which is typical.  The problem is
stationary phase cells have alot of rRNA and very little mRNA. 

I have considered labeling the total RNA directly (cDNA with aminoallyl
dUTP) but again I would have to scale up the cDNA reaction to over a 100ug
of starting total RNA (can you even do this). It is probable that 100ug of
total RNA would yield less than 100ng of cDNA for stationary cells.  I don't
think this is enough to label.

I don't feel there are alot of choices.  What I need to be able to do is get
out the mRNA with the least amount of rRNA as possible.  I have ordered up
an Ambion OligodT Magnetic bead kit.  There are other vendors out there that
have kits with 'special' ingredients they say will improve the recovery of
mRNA.  It is unclear whether any of these would work any better than the
things I have already tried. 

If anyone has any insight it would be welcome.

Thanks

Pat


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