Denaturation and electrophoresis of RNA with glyoxal

This protocol is used to denature and separate large mRNA molecules (0.5–10 kb) on agarose gels by electrophoretic size fractionation. Glyoxal (also called diformyl or ethanedial), the agent responsible for maintaining denaturation in this protocol, contains two carbonyl groups that react to form a cyclic ring structure with the imino and amino groups of guanine. It can also react with the amino groups of adenine and cytidine. When RNA is denatured in the presence of glyoxal, this covalent adduct prevents normal base pairing and maintains the RNA in a denatured state in agarose gels. Once formed, these adducts are stable at room temperature at pH