An siRNA molecule is formed from short complementary sense and anti-sense strands of RNA. The sense strand of the siRNA has the same sequence as a short region of the mRNA targeted for RNAi. Each siRNA strand may originate from different transcripts expressed in the same cell or both siRNA strands may be part of a single transcript that folds back to form a short hairpin structure [2] (Figure 1). The expression cassette(s) encoding the siRNA strands can be delivered to cells in the same fragment of DNA (hairpin expression cassette) or on separate fragments of DNA (sense and anti-sense strand expression cassettes; Figures 1 and 2).

Although PCR based siRNA expression cassettes have the advantages of being fast to produce and test, some users may choose to transfect the siRNA expression cassette in the form of a plasmid or to have a large supply of expression cassette DNA readily available. Any of the PCR products generated in the previous sections can be cloned into the siXpress(TM) Template if the target-specific downstream primer used to generate the PCR products include an Xho I or BamH I restriction site. For additional instruction on molecular biology procedures, refer to Sambrook et al.