High-throughput purification of double-stranded RNA molecules using convective interaction media monolithic anion exchange columns
| Autor: | Alesia Romanovskaya, Peter Sarin, Dennis Bamford, Minna Poranen, Alesia Levanova |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Small interfering RNA
Monolithic HPLC column 01 natural sciences Biochemistry Analytical Chemistry Bacteriophage 03 medical and health sciences chemistry.chemical_compound RNA interference RNA polymerase Anion Exchange Resins 030304 developmental biology RNA Double-Stranded 0303 health sciences Chromatography biology fungi 010401 analytical chemistry Organic Chemistry RNA General Medicine biology.organism_classification Chromatography Ion Exchange 0104 chemical sciences RNA silencing chemistry biology.protein Dicer |
| Zdroj: | Journal of chromatography. A. 1278 |
| ISSN: | 1873-3778 |
| Popis: | Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58-1810 bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25-27 bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved. |
| Databáze: | OpenAIRE |
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