Production of dsRNA for RNAi in Avian Embryos
This protocol was adapted from “RNAi in Avian Embryos,” contributed by Esther T. Stoeckli, Chapter 14, in RNAi: A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
This protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos.
