Small interfering RNAs (siRNAs) have emerged as a powerful technique for sequence-specific gene silencing in a wide variety of organisms. However, the base composition of the siRNA sequence is not the only determinant of efficacy. Intrinsic factors related to mRNA structures are likely to be crucial determinants for siRNA activity. Indeed, placing the recognition site of an active siRNA into a structured mRNA region has abrogated the siRNA activity. Therefore, a successful gene-targeting project may require the design of many distinct siRNAs at a high cost. Here, potential design rules, cost-effective strategies for producing siRNAs by T7 RNA polymerase, and expression cassettes for in vivo testing are described.
