Kinetic and Efficacy Analysis of RNA Interference in Stably and Transiently Expressing Cell Lines

RNA interference, particularly through the use of small interfering RNA (siRNA), has become an important laboratory tool for both fundamental and applied investigations. However, it is currently unknown whether siRNA-mediated knockdown of transiently expressed proteins is an acceptable quantitative surrogate for stably expressed proteins. Further, the best means by which to transfect cells with functionally active siRNA are poorly defined, and determination of the best reagent and transfection conditions for a particular cell line is a burdensome prerequisite for RNA interference studies. We therefore established the optimal transfection conditions for six commercial siRNA delivery reagents in three cell lines (HR5-CL11, HeLa, and NIH/3T3) transiently or stably expressing the firefly luciferase gene. The delivery efficiency, knockdown kinetics, and cytotoxicity of the reagents were evaluated. siPORT Amine, X-tremeGENE, and TransIT-siQUEST achieved the best knockdown and consistency of performance among the three cell lines. Delivery efficiency varied and was cell line dependent in some cases. The knockdown kinetics were reagent-dependent, and knockdown was generally more rapid in the stably transfected cells. Cytotoxicity of the reagents was variable. GeneSilencer was the least cytotoxic reagent for all three cell lines, and TransIT-siQUEST was the most cytotoxic to the HeLa and HR5-CL11 cell lines. These comparative results provide an initial basis for reagent selection and experimental design for RNA interference studies in HeLa, NIH/3T3, and their respective derivative cell lines. Keywords: Gene knockdown; luciferase; RNA interference; siRNA