Preparation of dsRNA for microinjection experiments in mouse.
RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. The protocol presented here is designed for the preparation of microgram amounts of double-stranded RNA (dsRNA) for microinjection experiments in mouse oocytes and early embryos. Briefly, dsRNA is produced after annealing sense and antisense RNA strands, or spontaneously during in vitro transcription of an inverted repeat. We usually include RNase T1 treatment of the annealed RNA prior to the purification step in order to remove unannealed single-stranded RNA (ssRNA), which can interfere with the quantification of dsRNA in a nondenaturing agarose gel.
Svoboda
Petr
P
Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. svobodap@img.cas.cz
D000818:Animals; D001483:Base Sequence; D005821:Genetic Techniques; D051379:Mice; D008845:Microinjections / Q000379:methods*; D008957:Models, Genetic; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D014158:Transcription, Genetic
D012330:RNA, Double-Stranded; D012313:RNA