Rapid purification of RNAs using fast performance liquid chromatography (FPLC) - PubMed (original) (raw)

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

Insil Kim et al. RNA. 2007 Feb.

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.

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Figures

FIGURE 1.

FIGURE 1.

Schematic outline of RNA sample preparation. Estimated time for each step is shown in parentheses. Desalting and size-exclusion columns are equilibrated before use.

FIGURE 2.

FIGURE 2.

Purification of HIV TAR RNA (60 nt) by size-exclusion chromatography. Elution profile obtained from the size-exclusion chromatography step on the Superdex 200 (26/60) column. FPLC was performed at 3 mL/min with 10 mM phosphate buffer (pH 6.5) and 100 mM NaCl.

FIGURE 3.

FIGURE 3.

RNA sample purity. (A) The secondary structure of the TAR RNA is shown. (B) Imino proton region of 1D 1H NMR spectra of HIV TAR RNA purified by size-exclusion chromatography. Assignments are indicated numerically above the peaks. (C) Denaturing 10% TBE gel of the NMR sample used in B, stained with toluidine blue.

FIGURE 4.

FIGURE 4.

Standard curve of double-stranded RNAs on (A) Superdex 200 (10/30) and (B) Superdex 75 (10/30) columns. FPLC was performed at 0.4 mL/min in 10 mM phosphate buffer (pH 6.5) and 100 mM NaCl.

FIGURE 5.

FIGURE 5.

Purification of the desired RNA product from hammerhead ribozyme by Superdex 200 (10/30) size-exclusion chromatography. (A) Elution profile obtained from the size-exclusion chromatography is shown. FPLC was performed at 0.5 mL/min with 10 mM phosphate buffer (pH 6.5) and 50 mM NaCl. (B) The purity of the eluted sample is shown by denaturing PAGE on 5% TBE gels. The elution volume is indicated at the top of each lane.

FIGURE 6.

FIGURE 6.

Purification of CpGAIT (34 nt) with Superdex 75 (26/60). Elution profile obtained from the size-exclusion chromatography step using the Superdex 75 (26/60) column. FPLC was performed at 3 mL/min with 10 mM phosphate buffer (pH 6.5) and 100 mM NaCl.

FIGURE 7.

FIGURE 7.

Renaturation of oligomerized RNA after storage. Superdex 200 (10/30) size-exclusion chromatography performed at 0.5 mL/min with 10 mM phosphate buffer (pH 6.5) and 50 mM NaCl. Chromatogram shows a 69mer RNA stored in −20°C freezer and thawed, without additional heating (top). Upon 90°C heating for 5 min and rapid cooling on ice, most of aggregation and dimerization is eliminated, as shown in chromatogram (bottom).

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