resolved and any degradation (seen as a smear) or DNA contamination is seen easily.
This can be achieved on a 2.55% acrylamide gradient gel with an overnight run.
Both these methods involve running native RNA. There will almost certainly be some
secondary structure within the RNA molecule owing to intramolecular hydrogen
bonding (see e.g. the clover leaf structure of tRNA, Fig. 5.6). For this reason native
RNA run on gels can be stained and visualised with ethidium bromide. However, if the
study objective is to determine RNA size by gel electrophoresis, then full denaturation
of the RNA is needed to prevent hydrogen bond formation within or even between
polynucleotides that will otherwise affect the electrophoretic mobility. There are three
denaturing agents (formaldehyde, glyoxal and methylmercuric hydroxide) that are
compatible with both RNA and agarose. Either one of these may be incorporated into
the agarose gel and electrophoresis buffer, and the sample is heat denatured in the
presence of the denaturant prior to electrophoresis. After heat denaturation, each of
these agents forms adducts with the amino groups of guanine and uracil, thereby
preventing hydrogen bond reformation at room temperature during electrophoresis.
It is also necessary to run denaturing gels if the RNA is to be blotted (northern blots,
Section 5.9.2) and probed, to ensure that the base sequence is available to the probe.
Denatured RNA stains only very weakly with ethidium bromide, so acridine orange is
commonly used to visualise RNA on denaturing gels. However, it should be noted that
many workers will be using radiolabelled RNA and will therefore identify bands by
autoradiography. An example of the electrophoresis of RNA is shown in Fig. 10.16.
10.5 Capillary electrophoresis
The technique has variously been referred to as high performance capillary electro-
phoresis (HPCE), capillary zone electrophoresis (CZE), free solution capillary electro-
phoresis (FSCE) and capillary electrophoresis (CE), but the term CE is the one most
25S–3396b
18S–1800b
5,8S–158b
5S–121b
tRNA_72b
1234
Fig. 10.16Separation of yeast RNA on a 1.5% agarose gel. (Courtesy of Dr Tomas Masek, Department of
Genetics and Microbiology, Charles University, Prague, Czech Republic.)
427 10.5 Capillary electrophoresis
