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.

FIGURE 1.

IP RP HPLC chromatogram of eukaryotic total RNA. The chromatogram shows the analysis of (3 μg) total RNA extracted from Hela cells using gradient 2 (see Materials and Methods). A shows the typical profile of the early eluting small RNA species and the later coeluting 18S and 23S rRNA. B shows an enhanced view of the smaller RNA species. The tRNA, 5.8S, and 5S rRNA are highlighted.

FIGURE 2.

Solid phase enrichment and fractionation of RNA using alkylated poly(styrene-divinylbenzene) particles. A shows the IP RP HPLC chromatogram of the enriched and fractionated large RNA species from total RNA extracts. B shows the IP RP HPLC chromatogram of the enriched small RNA species in the absence of larger rRNA species.

FIGURE 3.

IP RP μHPLC chromatogram of eukaryotic total RNA. The chromatogram shows the analysis of 9.2 ng total RNA extracted from Hela cells using gradient 4 (see Materials and Methods). A shows the typical profile of the early eluting small RNA species and the later coeluting 18S and 23S rRNA. B shows an enhanced view of the smaller RNA species. The tRNA, 5.8S, and 5S rRNA are highlighted.

FIGURE 4.

IP RP μHPLC chromatogram of the enriched small RNAs from total RNA spiked with synthetic let-7 miRNA. The chromatogram shows the analysis of the small RNA fraction obtained following the solid phase enrichment of a total RNA (20 μg) spiked with synthetic miRNA (let-7 150 ng) using gradient 5 (see Materials and Methods). The let-7 miRNA species is highlighted.