title: Measuring RNAi knockdown using qPCR.
abstract: We describe a scalable method for measuring changes in gene expression following RNAi in mammalian cells. This protocol outlines methods to transfect cells, isolate total RNA, perform cDNA synthesis, run qPCR reactions with multiplexed TaqMan dual hydrolysis probes, and analyze the results from qPCR using relative quantification (Fleige et al., 2006). These methods can be used to assess the relative knockdown of a set of siRNAs against a target in order to identify the most effective molecule and they can be used to assess the potency of a siRNA or group of siRNAs when performed over a range of doses. When screening panels of siRNAs targeting a given gene, knockdown can be assessed in two phases. In the first phase, the most active molecules are identified in a single dose screen. In the second phase a dose response screen is used to identify the subset of efficacious molecules that are the most potent.
authors.affiliation: Alnylam Pharmaceuticals, Cambridge, MA, USA.
authors.forename: Stuart
authors.initials: S
authors.lastname: Milstein
authors.forename: Michael
authors.initials: M
authors.lastname: Nguyen
authors.forename: Rachel
authors.initials: R
authors.lastname: Meyers
authors.forename: Antonin
authors.initials: A
authors.lastname: de Fougerolles
mesh_terms: D000818:Animals; D018076:DNA, Complementary; D055785:Gene Knockdown Techniques / Q000379:methods; D008322:Mammals; D012313:RNA / Q000302:isolation & purification; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D014162:Transfection / Q000379:methods
chemical_list: D018076:DNA, Complementary; D034741:RNA, Small Interfering; D012313:RNA
keywords: Branched deoxyribonucleic acid assay (bDNA); Fluorescence resonance energy transfer (FRET); Quantitative PCR (qPCR); RNA isolation using magmax™ magnetic bead purification; RNAi knockdown measurement; Ribonucleic acid (RNA); Transfection; cDNA synthesis