Transverse gradient gel electrophoresis: a gel system for resolving complex mixtures of lariat and linear RNA molecules.

In studying the complex RNA processing pathways of premRNAs with multiple introns it is often necessary to resolve RNA splicing intermediates and different topological forms of RNA. Conventional one dimension northern hybridization analysis does not offer enough resolution to discriminate intron lariats from larger unspliced pre-mRNAs. Historically, two gels with different concentrations of acrylamide have been used to distinguish between circular and linear RNA species (1, 2). However, these analyses were done on P-labelled, in vitro synthesized RNA subjected to in vitro splicing reactions where sensitivity of detection is not a problem and the number of RNA species are limited. Such an analysis on in vivo RNA of genes containing multiple introns is ambiguous due to the many linear and lariatcontaining splicing intermediates and the low concentrations of these intermediates in cellular RNA samples. Detecting lariat RNAs with 2 gels is further complicated with complex RNA mixtures when an RNA band detected on one gel cannot be equated with the comparable band on another gel because the RNAs change migration order. We have therefore developed a new method to discriminate between linear and lariat RNAs in a complex, cellular RNA sample. During transverse linear gradient polyacrylamide gel electrophoresis (TGGE) under denaturing conditions, the electrophoretic mobility of lariatcontaining RNA molecules changes much more dramatically as a function of polyacrylamide concentration than that of linear RNAs. In a 3.5-8% polyacrylamide TGGE, lariat molecules run as downward arcing bands that cross over linear molecules. This feature of TGGE allows one to infer relationships between linear and lariat-containing RNAs based on their band shape across the entire gel. TGGE also offers superior sensitivity of detection due to pattern recognition compared to a ID gel, because very faint signals that might be ignored as background in a ID gel appear as distinct arcs in TGGE.