RNA Electrophoresis

Electrophoresis through agarose or polyacrylamide gels is the standard way to separate, identify and purify nucleic acid fragments. The location of the nucleic acid within the gel can be determined by using the fluorescent intercalating dye ethidium bromide.

Agarose gels have a smaller resolving power than polyacrylamide gels but a greater range of separation - from 200 bp to >50 kb using standard gels and electrophoresis equipment. RNAs up to 10 000 kb can be separated in agarose gels using pulsed field gel electrophoresis.

Polyacrylamide gels have enough resolving power to separate fragments differing by only one base pair in size, but their range is ~ 5 to 1000 bp. They are much more difficult to handle than agarose gels.

Formaldehyde-agarose gel electrophoresis top

Agarose is a polysaccharide obtained from seaweed. There are frequently contaminants - other polysaccharides, salts and proteins - and different batches as well as different manufacturers brands vary in the level of contaminants and hence in the performance of the agarose. Agarose can be chemically modified to gel and melt at lower temperatures by the addition of hydroxyethyl groups into the polysaccharide chain.

Agarose gels are cast by completely melting the agarose in the desired buffer and then pouring into a mould to harden. RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode.

RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form

The rate of migration is determined by

Molecular size of RNA

Linear RNA becomes orientated in an electric field in an 'end-on' position and migrates through the matrix of the gel at a rate which is inversely proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly because of greater frictional drag as they try to pass through the gel matrix.

Agarose concentration

Linear RNA of a given size migrates through agarose of different concentrations at different rates given by log m = logmo -KrT, where m is the electrophoretic mobility of the RNA, mo is the free electrophoretic mobility of the RNA, Kr is the retardation coefficient and T is the gel concentration

Conformation of the RNA

RNA molecules which fully or partially retain their secondary structure, migrate at different rates to fully denatured RNAs with the same molecular mass

Applied voltage.

At low voltage, the rate of migration of RNA is proportional to the voltage applied. As the voltage is increased, the mobility of larger molecules increases differentially: the effective range of separation therefore decreases with increasing voltage. For maximum resolution, run agarose gels at no more than 5V / cm (measured between the electrodes, not the gel length).

Base composition of the RNA and temperature of the gel

For agarose, neither of these parameters significantly affect the mobilities of RNA

Presence of intercalating dyes

Ethidium bromide reduces the electrophoretic mobility of linear RNA by about 15%. EtBr has greater affinity for double than single-stranded nucleic acids.