Enrichment and analysis of RNA centered on ion pair reverse phase methodology

Abstract

Here we describe a procedure for the rapid enrichment of RNA from cell extracts and the subsequent fractionation and analysis of the “small RNA” population by ion pair reverse phase chromatography. Solid phase extraction procedures have been developed utilizing nonporous alkylated poly(styrene-divinylbenzene) particles in conjunction with ion pair reagents to enrich total RNA. This approach facilitates the selective enrichment and separation of the relatively lower abundance small RNAs, from the more abundant higher molecular weight rRNA species. We also describe the application of monolithic capillaries in conjunction with ion pair reverse phase chromatography to bring increased sensitivity in the analysis of very low abundance RNAs. These approaches will simplify the biochemical analysis of this class of molecules, which are emerging as important regulators of global gene expression in higher organisms.

Keywords

• ion pair reverse phase chromatography
• solid phase extraction
• total RNA
• small RNAs and miRNA