RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos.  The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform.  In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol]

Protocol describes a method to prepare and fractionate Drosophila embryo extracts.  These extracts can be used for the in vitro characterization of RNAi. - [Read Preparation and Fractionation of Drosophila Embryo Extracts for the In Vitro Characterization of RNA]

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Protocol describes how to prepare double-stranded RNA (dsRNA) for RNA interference in Drosophila by synthesis of individual RNA strands from linearized plasmid templates, followed by annealing of the strands.  DsRNA molecules with a length of 500-800 bp seem to be most active.  The dsRNA can be made from cDNA or genomic DNA templates, as long as most of the dsRNA corresponds to presumptive exon sequence. - [Read Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method Protocol]