RNA sizes

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Mon Jun 2 23:15:46 EST 1997


David F. Spencer (dspencer at is.dal.ca) wrote:

: In article <338C0991.1E9D at ukbf.fu-berlin.de>, schmidt-ott at ukbf.fu-berlin.de
: wrote:

: > Hello!  : > I made a weird observation concerning RNA sizes: I ran
total RNA : > from tissue cultures on a non-denaturating agarose gel to
check for : > integrity, using a DNA size marker (100 bp-Marker). On
repetition of the : > experiment, the 18S and 28S bands appeared in
different positions : > relative to the DNA marker. I suspect that this
might be due to : > different concentrations of agarose in the gel. Has
anyone made a : > similar observation ?  : > Secondly, I am desperately
looking for a source where I can find the : > exact bp sizes of the 18S
and 28S RNA bands in different species, for : > example in rats, mice,
cattle etc. 


: The mobility of 18S and 28S RNAs on non-denaturing gels will have no
particular : relationship to their actual sizes. These RNAs have
considerable secondary : structure and this is responsible for the
mobility differences between gels : of different pore sizes. In addition,
evaluating RNA quality on non-denaturing : gels will give unreliable
estimates of RNA degradation because secondary : structure can hold
together pieces that are no longer continuous. You should use : denaturing
agarose (formaldehyde) or acrylamide (urea) if that is the goal; the :
latter is the more practical for RNA's that are about 2 k or shorter and
much : easier to work with. For larger RNAs you're pretty much stuck with
either : formaldehyde, gloxal or methylmercury (yuk) agarose gels. 


I combined two Biotechniques articles to make a really reliable and fairly
simple protocol for good RNA gels.  Biotechniques 14:380 (1993) tells how
using 0.22 M (0.66%) formaldehyde in both gel and buffer (MOPS) keeps the
RNA denatured and running as nice tight bands (e.g. rRNA).  Biotechniques
14:932 (1993) tells how using 10ug/ml EtBr directly in the loading dye ,
with brief heating prior to loading, allows easy visualization and
efficient transfer (if blotting is to follow).  The amount of formaldehyde
used in this protocol is far less than usual.  See the article for
formulas.

Some of my colleagues here just run the RNA on plain agarose gels and
stain afterwards, but the RNA never looks as nice IMO, and it takes loner 
to visualize.





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