Identification of viroids by gel electrophoresis

Two-dimensional PAGE involving an initial fractionation under nondenaturing conditions followed by a second electrophoresis under denaturing conditions provides a powerful means to detect viroids and other small circular RNAs. This unit describes a method known as "R(eturn) PAGE" in which denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength during the second electrophoresis. Under denaturing conditions, circular RNAs migrate more slowly than their corresponding linear forms. Following fractionation, RNAs are visualized by staining with ethidium bromide, SYBR Gold, or silver nitrate. Unlike nucleic acid hybridization or RT-PCR, viroid identification by R-PAGE requires no nucleotide sequence information.