Northern blotting

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Jul 24 10:01:52 EST 1995


In Article <3urpas$54e at gazette.bcm.tmc.edu>, mtuvin at bcm.tmc.edu (Michael J.
Tuvin) wrote:
>In article <3ue8nn$f8l at azure.acsu.buffalo.edu> v115t2vq at ubvms.cc.buffalo.edu
writes:
>>A straight and simple question:
>>Is there any probe better than rRNA/actins in Northern blot for internal 
>>control?
>>Thank you!
>>Larry Lu
>>Dept. of Biochem. Pharmac.
>>SUNY at Buffalo
>>V115T2VQ at ubvms.cc.buffalo.edu

>Sure. cRNA/GAPDH or cDNA/GAPDH. Glyceraldehyde 3-Phosphate Dehydrogenase is
>less abundant and more uniform then actin.
>We by our probe from Clontech and very happy with it. 
>---------------------------------------------------------------------------
>Michael J. Tuvim 
>Research Assistant Professor, Dept.of Medicine, Baylor College of Medicine.
>Tel: 713/794-7794(5), FAX: 713/794-7853, e-mail: mtuvin at bcm.tmc.edu
>---------------------------------------------------------------------------

The best internal standard to use for RNA analyses really depends on what
type of treatment is being done in the experiment and how that treatment
affects the levels of the internal standard. Ideally, one does not want
their experimental treatment to alter the levels of the mRNA being used as
the internal standard. In practice it can sometimes be difficult to find a
standard such as this, but if you really want quantitative results you need
to take the time to test and confirm this. Numerous investigators have shown
that beta-actin mRNA levels vary in certain tissues and after certain
experimental treatments. The same is true for GAPDH. In fact, I think this
is potentially a really BAD internal control for most experiments, since
GAPDH levels also vary with developmental stage, as well as in various
tissues and with experimental treatments. Furthermore, many different
GAPDH-related sequences have been identified and there appear to be at least
300 copies of these in the rat genome and 100 copies in the human genome. To
make matters even worse, some of these pseudogenes are actually transcribed.
(NAR 13:7, 2485-2502). This does not make for an ideal internal control
probe! Having said all that, I'd suggest using a rRNA as the internal
standard, even if it's a pain. Since rRNA makes up approximately 90% of the
total RNA, even if its levels did vary some, it would still probably be the
best internal control for most experiments. The only way to be certain is to
convince yourself that the standard you are using is not affected by your
experimental treatment. I think it's best to compare standards before
beginning a long series of experiments, unless it's not important for your
data to be quantitative. Good luck.
    tracy



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