Chapter 13 - Electrophoresis of RNA

Abstract

Electrophoresis is a chromatography technique by which a mixture of charged molecules is separated according to size when placed in an electric field. The accurate determination of the size of RNA species is just as important as deduction of the molecular weight of any other macromolecules subjected to electrophoresis. This is accomplished by comparing the electrophoresis of an RNA sample against molecular weight standards. The successful electrophoresis of RNA is accomplished in two parts: RNA is denatured prior to loading the gel; and during the electrophoresis, conditions that support and maintain the denatured state are established. These steps will ensure that like species of RNA co-migrate, and the best possible resolution is achieved when gels are poured thinly and run at low voltage. The choice of denaturing system and gel matrix is determined primarily by the size range of the RNAs to be separated and the most commonly used RNA denaturants for agarose gel electrophoresis are formaldehyde and urea. Several gel staining techniques are used for the visualization of nucleic acids in agarose gels such as ethidium bromide, SYBR green, SYBR gold, SYBR safe, GelStar, silver staining, acridine orange, and methylene blue. The proper maintenance and use of electrophoresis gel boxes and power supplies is of utmost importance for the safety of all.