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.

Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography

“…Using ion-pair, reversed-phase HPLC, previous studies show that RNAs can be separated under non-denaturing, partially denaturing, or fully denaturing conditions [23; 28]. We believe that the separation method we present here is a partially denaturing protocol despite the fact that we used no Mg 2+ ions and our separation was at elevated temperature, both of which correlated to more of a denatured condition.…”

“…The latter technique is less time consuming, and is more suitable for high throughput analysis [20]. To date, ion-pair, reverse-phase HPLC is a standard platform for separation and quantification of a wide range of labeled/unlabeled, regular/modified RNAs [23], such as siRNAs [24; 25; 26], mRNA [27], and large ribosomal RNA (rRNA) [28]. However, HPLC can be used to separate short RNAs (~20 nt) by single-nucleotide resolution [29; 30; 31].…”

A multidimensional platform for the purification of non-coding RNA species

“…The multidimensional chromatography platform developed here also has advantages over existing chromatographic methods that target individual ncRNA species (18,22,32,50). The newer Agilent SEC columns solve difficulties with the separation of large molecular weight rRNA observed in earlier work with RNA SEC (22,32).…”

“…The newer Agilent SEC columns solve difficulties with the separation of large molecular weight rRNA observed in earlier work with RNA SEC (22,32). This limitation was only partially relieved in more recent efforts that use IP RPC for RNA separation (18,50). Conversely, purification of RNA species in the low MW size ranges (<100 nt) often requires an additional enrichment step either through the use of commercially available small RNA kits or with solid-phase extraction processes because of their low natural abundance and inefficiencies in extracting them with the commonly used acidic phenol chloroform method (51,52) without further enrichment on silica gel or glass fiber matrices (53).…”

Chromatographic methods for the determination of therapeutic oligonucleotides

“…In an excellent application for miRNAs, Dickman and Hornby used a commercially available capillary column with a TEAA mobile phase to concentrate miRNA from total RNA extracted from HeLa cells [77]. Let-7 miRNA was spiked into total extracted RNA.…”