mRNA isolation fron bacteria

Daniel Mytelka;612 Barker;x2-5110 mytelka at codon1
Tue Feb 9 16:31:31 EST 1993


In article <9FEB199307075942 at aardvark.ucs.uoknor.edu> broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
>In article <CMM.0.88.728441416.malam at uhunix.uhcc.Hawaii.Edu>, malam at UHUNIX.UHCC.HAWAII.EDU (Maqsudul Alam) writes...
>>Can anybody help us to isolate mRNA from bacterial cells? What is the best
>>method or kit available in the market?
>> 
>>Thanks.
>
>Because bacterial transcription and translation are coupled, stable
>mRNA will not be produced as it is in Eucaryote cells.  Thus, trying
>to isolate mRNA from bacterial cells is difficult if not next to impossible.
>There is no polyA tract on the 3' end of the bacterial mRNA so polyT
>columns are out.  Most folks deal with the gene directly and clone it
>from the bacterial DNA.


This answer is quite inaccurate. Purifying RNA from bacteria is quite
simple, and takes a couple of hours or so. If care is taken to make sure
that any RNAses present are inactivated, the RNA will be stable for a 
long while (the oldest RNA's I'm currently using are almost two years old).
While it's true that the lack of poly-A on bacterial RNA makes it
difficult to separate mRNA from tRNA and rRNA, the is usually not very
important. Primer Extension, SI, and other methods can easily be
employed on the mix. Obviously, cloning out the bacterial DNA is also
readily done, but that is useless for any quantitation of expression,
promoter mapping, etc (unless you want to make major assumptions about
in vivo vs. in vitro promoter usage).

The technique I use for Gm- cells is found in Current Protocols in
Molecular Biology (Ausubel et al), and basically is a lysis step with
lysosyme and SDS, followed by inactivation of RNAses with DEPC and
then precipitation of the RNA. There is a similar technique also there
for Gm+ cells, which has also been used successfully by others in this
laboratory.

Daniel Mytelka, mytelka at mendel.berkeley.edu
Dept of Molecular and Cell Biology
UC Berkeley



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