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RTPCR and RNA preps

Teresa Golden tgolden at arlab.biology.rochester.edu
Tue May 27 21:51:35 EST 1997


Hi all,

In regard to my questions about RNA preparation for RTPCR I received
several helpful responses that I was asked to pass on to the newsgroup.

Many suggested that despite the cost the Qiagen RNAeasy kit worked well in
preparing RNA for RTPCR with the caveat that some genomic DNA comes
through so it is wise to use DNase.  Others stated that the standard
phenol and guanidinium thiocyanate worked just fine.  Listed below are the
specific responses...

Thank you all for your suggestions,
      -Teresa

---
I=B4d like to refer you to a lab in Hamburg, Germany where a nice technique
for single cells (or a few cells, they usually use protoplased cells, but
it should be feasible from a few intact cells as well):
The person I know there is Margret Sauter (fb5a084 at botanik.uni-hamburg.de),
the technique was developed by Thomas Dresselhaus.

Dr. Tony Schaeffner
Institut f. Biochemie
Ludwig-Maximilians-Universitaet Muenchen
=46eodor-Lynen-Str. 25
D-81377 Muenchen
Germany

=46ax    +49-89-7401-7314
phone  +49-89-7401-7311
email  schaeff at lmb.uni-muenchen.de
----
We use the Quiagen RNAeasy kit for plant material, and minishredder (who
dreams up these product names?), and it works fine for RT PCR without a
poly-A enrichment step. It is worth the cost if time is the limiting factor;
if money limits (as it now does for me after using these kits), then its
back to the hot phenol.

John G. Turner
School of Biological Sciences
University of East Anglia
Norwich NR4 7TJ
England

--------
   We just prepare our samples in guanidinium thiocyanate &
phenol - that gives us great RNA.  I can send you the prep if you
need it, but I bet it's a lot like the one you're currently using.

>>note: she has kindly sent me the prep which I would be happy to forward
to anyone who needs it (just give me a few days). -Teresa<<

As for RNase inhibitors, the combo of guanidinium & phenol serve
to take care of RNases in the prep.  We use the perkin-elmer kit
for RT-pcr.  Incredibly expensive, but it's highly reproducible &
I didn't find that to be true with most other kits we tried.
The PE kit comes with an RNase inhibitor.  You need to add that
*first* to your RNA because, as PE freely admits, the reagents in
their kits are not RNase free (!).  I tested it & it's true - if
I add reagents before the inhibitor, I get no RT-pcr products
but if the inhibitor goes in first, everything is fine.

Margaret Hollingsworth
Bio Sci Dept, SUNY at Buffalo

----
I used both phenol extraction and GuHCl, both worked. I did not try to
do rigorously quantitative RT PCR, but you can spike 1st strand
synthesis with r/adNTP to normalize the cDNA yield, it works.

Dmitry A. Belostotsky
Assistant Professor

Department of Biological Sciences
State University of New York at Albany
1400 Washington Ave.
Albany NY 12222
Tel. (518) 442 4368
FAX  (518) 442 4767
E-mail dab at cnsvax.albany.edu
http://barbara.bio.albany.edu/~dab/

-------
 try Qiagen RNeasy plant mini prep. It has worked well for us on
     carrots, tobacco, corn, etc.

     Antonio Reinero
     Plant Biotechnology
     American Cyanamid

------
 We use plant RNEasy preps from Qiagen for RT-PCR.  Works fine but we
get some genomic sometimes so if you are using a lot of cDNA for your
PCR it is worth DNasing the RNA beforehand.  We don't use RNase
inhibitors as we never have problems with degradation.  If you use the
Qiagen preps you should have any problems of that sort.

================================================
Marc R Knight
Department of Plant Sciences,
University of Oxford,
South Parks Road,
Oxford OX1 3RB.
Tel. + 44 1865 275023
Fax. + 44 1865 275074
HomePage URL: http://users.ox.ac.uk/~dops0009
=============================================
-------
We have successfully used the "RNeasy plant total RNA prep" from QIAGEN to
obtain RNA from small amounts of Arabidopsis tissue. There is some genomic
DNA carry over, so I'd recommend treatment with RNase-free DNase before RT.

Also, the kit works great for Arabidopsis tissue, but we had trouble
isolating RNA from pea shoot apices as we had too much genomic DNA clogging
up the column. I don't remember the exact modification the company
suggested, but I think it involved diluting the homogenate further before
applying it to the column. It worked okay as a more dilute solution. You
may need to optimize the conditions if you're using tissue with small cells
and relatively high genomic DNA levels.

Mary Williams
Asst. Prof.
Biology Dept.
Harvey Mudd College
Claremont, CA

--------
I will reccomend isolating RNA using the protocol of Napoli et al
(1990). For RT-PCR see Metzlaff et al (1997), Cell, Vol88:845-854.
Michael Metzlaff does RT-PCR using small amount of tissues and I will
highly reccomend his method. See experimental procedure section of the
Cell paper. The reference of Napoli et al is also mentioned in his
paper. Alternatively you can contact him at the John Innes Centre by
email; metzlaff at bbsrc.ac.uk (although it has been bouncing lately) or
fax. Fax no. is (1603)-456844. Phone: (1603)-452571/ext 2279. I hope
this helps.
Manash Chatterjee

-----






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