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Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel PCR strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs in any vector. IR-PCR relies on differentially tagging antisense and sense copies of the target in one round of PCR and assembling them in a second. We used IR-PCR to assemble constructs targeting profilin, actin, and actin-related protein (ARP) transcripts from Arabidopsis. Immunoblotting of lines expressing a profilin PRF1 3' untranslated region (UTR)-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.

Inverted Repeat PCR for the Rapid Assembly of Constructs to Induce RNA Interference