User`s guide

Contents ▲ 57 ▼ Index
RNA are electrophoretically driven by a voltage gradient—similar to slab gel
electrophoresis. Because of a constant mass-to-charge ratio and the presence of a
sieving polymer matrix, the molecules are separated by size. Smaller fragments are
migrating faster than larger ones. Dye molecules intercalate into DNA or RNA strands or
Protein-SDS micells. These complexes are detected by laser-induced fluorescence. Data
is translated into gel-like images (bands) and electropherograms (peaks). With the help
of a ladder that contains fragments of known sizes and concentrations, a standard curve
of migration time versus fragments size is plotted. From the migration times measured for
each fragment in the sample, the size is calculated. Two marker fragments (for RNA only
one marker fragment) are run with each of the samples bracketing the overall sizing
range. The “lower” and “upper” markers are internal standards used to align the ladder
data with data from the sample wells. This is necessary to compensate for drift effects
that may occur during the course of a chip run.
For DNA and protein assays, quantitation is done with the help of the upper marker. The
area under the upper marker peak is compared with the sample peak areas. Because the
concentration of the upper marker is known, the concentration for each sample can be
calculated. Besides this relative quantitation, an absolute quantitation is available for
protein assays, using external standard proteins.
For RNA assays, quantitation is done with the help of the ladder area. The area under the
ladder is compared with the sum of the sample peak areas. The area under the “lower”
marker is not taken into consideration. For total RNA assays, the ribosomal ratio is
determined, giving an indication on the integrity of the RNA sample.