Polyacrylamide gel electrophoresis of RNA

Abstract

Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to </=20 nucleotides (nt). In certain circumstances, e.g., resolving different conformers of RNAs or RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (> or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.