title: Optimization of transfection conditions and analysis of siRNA potency using real-time PCR.
abstract: RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.
authors: Molecular and Cell Biology Division, Life Technologies, Austin, TX 78744-1832, USA. angie.cheng@lifetech.com
authors: Angie
authors: A
authors: Cheng
authors: Susan
authors: S
authors: Magdaleno
authors: Alexander V
authors: AV
authors: Vlassov
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D005260:Female; D055785:Gene Knockdown Techniques / Q000379:methods*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D034622:RNA Interference / Q000187:drug effects; D020871:RNA Stability; D012333:RNA, Messenger / Q000037:antagonists & inhibitors* / Q000235:genetics; D034741:RNA, Small Interfering / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection
chemical_list: D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)