Effective Concentrations and Effectiveness of siRNA

RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.

RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.

HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.

Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.