Total RNA extraction...

David L Haviland David.L.Haviland at uth.tmc.edu
Tue Oct 10 08:49:59 EST 2000


kyungho1 at snu.ac.kr wrote:

> Hello. everyone...
> I have been doing Total RNA extraction out of bovine muscle and fat
> tissues. I couldn't get pure
> band size...I tried several different methods to get accurate Total RNA
> using TRIzol regent and
> Guanidium method. I still didn't get it yet...
>
> When I got samples from cattle, I put samples into 50ml falcon tube.
> Then tissues directly in nitrogen tank.
> Trizol regent 10ml per musscle 1g.
> Homogenizing with electric homogenizer(IKA homogenizer)
> transfer samples into 1.7ml tubes. 1ml/tube
> add 0.2ml chloroform -> centrifuge 12,000g 15 min. 4 cel degree
> transfer aquueous phaese to new tube, add 0.5 ml isopropyl alcohol ->
> 12,000g 10 min. 4 cel
> degree
> remove supernate. add 1ml 75% ethanol -> vortexing -> 7,500g 5 min. 4
> cel degree
> remove supernate. vacum dry 5 min.
> dissolving the RNA with DEPC water 80ul.
> actually I followed the same procedure from Life technologies
>
> Electrophoresis.(6.5% formaldehyde composition)
> 1% agarose gel.
> DEPC 58.0 ml
> heating -> cooling to 50-60 cel degree
> add 10X MOPS 8 ml
> add Formaldehyde 14.0 ml -> cooling
>
> Formamide 5 ul
> 10X MOPS 2 ul
> Formaldehyde 1.8 ul
> EtBr 1 ul
> add Sample 9.2 ul -> 65 cel degree heating for 15 min. -> ice 3 min
> add loading buffer 1 ul.
> loading into gel... 60 voltage about 2 hr
>
> That is all my protocols for my experiment.
> Do you have any suggestion about my protocol.
> I really need someone's help and advice. I had been stop this process
> for 2 months.
> Hope to hear from you...
> Have a wonderful day...

Hi:

This may not be what you want to hear but in my experience, these type of
tissues need to be minced under LN2.  We use a mortar and pestle that is
precooled in a bucket with LN2.  Once the tissue is ground into a fine
powder GTC is immediately added.  (Mortar and pestle are autoclaved before
use - BTW).   To tear up the tissue and the HMW DNA, the mix is taken
through a series of needles in decreasing size - 18, 20, 23 guage and it
continues until single drops emerge from the needle tip.   From this point
you can continue with the Chom & Sacchi protocol or personally since this
much effort has been invested, I'd go with  Chirgwin and take the solution
over 7.5M CsCl  and a SW50 rotor (or equivalent).  See Maniatis for it all.

Hope this helps,
David
===========
David L. Haviland, Ph.D., Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd.,  Houston, TX  77030
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously
overlooked something.
==========



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