A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics

Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription real-time polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-basepair overlapping RT primer facilitated the distinction of full-length antisense from its 3’ metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RISC-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing of the in vitro-in vivo translation of siRNA therapeutics.