| Custom RNA |
| RNA Purification |
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After synthesis, the crude RNA oligo is analyzed by HPLC and the solvent removed in preparation for shipping. There is no additional processing between synthesis and shipping. A number of researchers have reported that for many applications the crude RNA is sufficiently pure to use without further purification (Please note that the crude RNA is of as high or higher quality than DNA oligos). Desalting or purification is recommended if greater homogeneity is desired or considered necessary. The RNA oligos can be purified by either PAGE or anion exchange HPLC. Both the 2'-protected or 2'-hydroxyl RNA can be purified via PAGE or anion exchange HPLC.
PAGE-purification of RNA oligonucleotides when fully deprotected requires no modification of standard PAGE protocols, although sterile conditions need to be implemented when appropriate. (For example, Maniatis T, Fritsch EF, and Sambrook J, Molecular Cloning, Cold Spring Harbor Press, 1982, p 173) Purification of the RNA when 2'-ACE protected requires minor modifications of standard PAGE protocols as the 2'-protecting groups are slowly hydrolyzed at pH 8.0 at high temperatures, i.e. > 50°C. These are conditions that the RNA may be subjected to during standard PAGE purification. Dharmacon has optimized PAGE protocols for purifying 2'-protected RNA.
Dharmacon offers PAGE purification services as an additional option. RNA oligonucleotides are purified in the stable 2'-protected form on 45 cm denaturing gels to provide excellent separation of the main product band. The product band is excised from the gel, the RNA eluted, the eluant filtered and desalted via reversed-phase cartridges. The purified RNA is shipped in the 2'-protected form to best ensure reliable purity. The pure RNA is deprotected just prior to use using the volatile buffer system to yield pure 2'-hydroxyl RNA. (Note: No degradation of pure 2'-OH RNA is observed under standard 2'-deprotection conditions.) The salts and by-products of the 2'-deprotection are all volatile and are readily removed along with the water by lyophilization or by use of a Speed-Vac. Please refer to Technical Bulletin #001 for further details on the 2'-deprotection system.
HPLC-purification of the RNA oligonucleotides is usually best effected using anion exchange chromatography. Either the 2'-protected or 2'-deprotected forms are suitable. The 2'-protected form offers the advantage of minimizing secondary structure effects and providing resistance to nucleases. The fully deprotected RNA requires sterile conditions during purification. Please refer to HPLC Results for suggested anion exchange HPLC conditions.
Historically, HPLC has often been used to purify RNA oligos because older chemistries yielded contaminating side products that are full length but are chemically modified, e.g. residual 2'-O-silyl groups. These contaminating products were best removed via HPLC. 2'-ACE chemistry yields a significantly cleaner product. In the development of purification services, no increased purity was observed with HPLC purification compared to PAGE purification. All PAGE-purified oligos are assayed by anion exchange HPLC and are greater than 97% pure by HPLC. |
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