Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

RNA interference (RNAi) is a promising new approach for oncogene targeting for “undruggable” targets, including KRAS. Here, we present a protocol for evaluating mutant selectivity of KRAS small interfering RNAs (siRNAs) using orthogonal in vitro and in vivo techniques. We describe steps for structural analyses of siRNA complexes, utilization of isogenic HA- and luciferase-tagged cell lines, RNA sequencing for off-target effects, and in vivo evaluation of mutant selectivity. This protocol has potential application for the development of mutant-specific siRNA molecules targeting any oncogene. For complete details on the use and execution of this protocol, please refer to Stanland et al. 1 • Procedure for evaluation of thermodynamic binding of siRNA to target mRNA • Steps for in vitro analysis of siRNA by RT-qPCR, western blotting, and luciferase assays • Instructions for in vitro analysis of off-target effects of siRNA using RNA sequencing • Steps for in vivo analysis of siRNA targeting by RT-qPCR in tumor and somatic tissues Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. RNA interference (RNAi) is a promising new approach for oncogene targeting for “undruggable” targets, including KRAS. Here, we present a protocol for evaluating mutant selectivity of KRAS small interfering RNAs (siRNAs) using orthogonal in vitro and in vivo techniques. We describe steps for structural analyses of siRNA complexes, utilization of isogenic HA- and luciferase-tagged cell lines, RNA sequencing for off-target effects, and in vivo evaluation of mutant selectivity. This protocol has potential application for the development of mutant-specific siRNA molecules targeting any oncogene.