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 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.
Kim
Insil
I
Department of Structural Biology, Stanford Magnetic Resonance Laboratory, School of Medicine, Stanford University, CA 94305, USA.
McKenna
Sean A
SA
Viani Puglisi
Elisabetta
E
Puglisi
Joseph D
JD
D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016337:RNA, Catalytic / Q000302:isolation & purification
D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D012313:RNA