Separation of oligo-RNA by reverse-phase HPLC

Gloria Mcfarland, Philip N. Borer

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

A rapid and highly reproducible chromatographic technique has been developed for analysis and purification of complex mixtures of oligoribonucleotides. The method utilizes a column of microparticulate porous silica beads fully derivatized with octadecylsilyl groups. The column is eluted with gradients in acetonitrile/water/anmonium acetate pumped at pressures of 1500-3000 psi. Most separations are completed in 5-15 min. with usually better than 1% reproducibility of absolute retention times and about 0.1% reproducibility of relative retention times. A single column accomplishes separations of menonucleosides, mononucleotides, and larger oligomers through at least 20-mers. The absolute detection limit is ∼1 pmole of base though most of the analytical separations described use ∼1 nmole. In favorable circumstances it is possible to use the analytical columns to purify ∼1 mg of an oligonucleotide in a single 10-30 min. elution.

Original languageEnglish (US)
Pages (from-to)1067-1080
Number of pages14
JournalNucleic acids research
Volume7
Issue number4
DOIs
StatePublished - Oct 25 1979

ASJC Scopus subject areas

  • Genetics

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