mRNA isolation without DNA!

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Wed Feb 9 10:44:31 EST 2000


In article <389fdefe at redeye.it.ki.se>, "Csaba Kiss"
<csakis at ki.se>
wrote:
>Hi guys!
>I would like to do subtractive hybridization on cDNA. I want to
isolate
>mRNA, which is free from even traces of DNA. The acidic phenol
method is the
>generally used method to isolate total RNA, and it is based on
the fact that
>the DNA will remain in the  phenolic phase if the phenol's pH
is acidic (ph
>5.0 ?). Then people do mRNA separation on oligo-T beads or
columns.  I have
>a feeling that these separations do not get rid of your
residual DNA
>totally. There is another way of isolating cytoplasmic RNA. You
can gently
>lyse the cells in a way that the nuclei remain intact. You
pellet down the
>nuclei, and work with the supernatant. This way you got rid of
all the
>genomic DNA. However, in such a mild lysis buffer there is no
Rnase
>inhibitor. How would I prevent my mRNA being degraded by the
endogenous
>Rnase? Dynal recommends to work quickly on ice. I have a gut
feeling that
>this does not eliminate RNA degradation. What do you think?
>
>Csaba
>csakis at ki.se

For most cell lines that I have worked with, the RNA is fine
using
this method even without the addition of an RNase inhibitor. I've
also run a lot of polysome gradients in the past using similar
procedures without noticable degradation of RNA.

My "gut feeling" is that most intracellular nucleases that could
be a problem are in the nucleus, so that if you don't disrupt the
nucleus, you are all right. No data, just a feeling.

Try lysing using a hyptonic solution with a gentle detergent:

10 mM NaCl or KCl,
3-5 mM MgCl2 (to keep nucleus intact),
10mM Tris or HEPES, pH 7.4 or so,
0.5% Nonidet P-40 or Triton X-100

Lyse and spin in the cold; to the super., add EDTA to 5-10 mM,
SDS
to 0.5%-1%, Proteinase K to 50-100 ug/ml. Digest at 37-45C for
39-
60 min., add salt to 100 mM, phenol/chloroform extract, EtOH
ppt.,
etc.

I used to do this when I needed to compare cytoplasmic vs. total
RNA.


Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu

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