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. 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).

PCR Expression Cassettes can be cloned using either the Hind III or EcoR I site upstream of the promoter and Xho I or BamH I site downstream of the siRNA and termination sequence. The cloning sites used must be unique in the PCR product. Hind III and Xho I are recommended because these restriction enzymes function with compatible buffers.