Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

Insil Kim; Sean A. McKenna; Elisabetta Viani Puglisi; Joseph D. Puglisi

doi:10.1261/rna.342607

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

¹Department of Structural Biology, School of Medicine, Stanford University, Stanford, California 94305, USA
²Stanford Magnetic Resonance Laboratory, School of Medicine, Stanford University, Stanford, California 94305, USA

Abstract

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.

Keywords

Footnotes

Reprint requests to: Joseph D. Puglisi, Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA 94305, USA; e-mail: puglisi{at}stanford.edu; fax: (650) 723-8464.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.342607.
- Received October 10, 2006.
- Accepted November 8, 2006.