Strategy and experimental verification for screening effective siRNA probes using target-reporter fusion. (A) Reporter gene fused at either C-terminal or (B) N-terminal of target gene. A panel of siRNAs or shRNAs against target gene is shown (▴). Efficacy of siRNA-mediated target gene silencing was measured by quantitation of reporter gene expression. C2C12 cells were cotransfected with either EGFP-specific siRNA (upper panel) or nonspecific siRNA (lower panel) with (C) two independent plasmids expressing EGFP and RFP, (D) a plasmid expressing EGFP-RFP fusion protein, and (E) a plasmid expressing RFP-EGFP fusion protein. DAPI, 4′, 6′-diamidino-2-phenylindole hydrochloride; EGFP, enhanced green fluorescent protein; RFP, red fluorescent protein; NON-SP, nonspecific siRNA; SP, specific siRNA. A scrambled siRNA served as a nonspecific control.
