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

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 oligonucle...

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Bibliographic Details
Published inRNA (Cambridge) Vol. 13; no. 2; pp. 289 - 294
Main Authors Kim, Insil, McKenna, Sean A, Viani Puglisi, Elisabetta, Puglisi, Joseph D
Format Journal Article
LanguageEnglish
Published United States Cold Spring Harbor Laboratory Press 01.02.2007
Subjects
Chromatography, Gel - methods
Chromatography, High Pressure Liquid - methods
Electrophoresis, Polyacrylamide Gel
Method
Oligodeoxyribonucleotides - chemistry
Oligodeoxyribonucleotides - isolation & purification
Oligodeoxyribonucleotides - metabolism
RNA - chemistry
RNA - isolation & purification
RNA - metabolism
RNA, Catalytic - isolation & purification
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Summary: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.
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Reprint requests to: Joseph D. Puglisi, Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA 94305, USA; e-mail: puglisi@stanford.edu; fax: (650) 723-8464.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.342607