ArticleBiology methods & protocols2024
A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics.
Article in Biology methods & protocols, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Pharmacokinetic/Pharmacodynamic Translation and Model-Informed Drug Development for Oligonucleotide Therapeutics.Journal of clinical pharmacology · 2026Review
- Staufen dsRNA-binding domain as modules to design bifunctional antibodies for siRNA delivery.Nucleic acids research · 2026Article
- Silencing of lncRNA HOTAIR Using Eyedrops as a Potential Treatment for Diabetes-Associated Retinal Dysregulation and Dysfunction.Investigative ophthalmology & visual science · 2026Article
- Advances in biomarkers for diagnosing and prognosticating disorders of consciousness.Frontiers in neuroscience · 2026Review
- Design of bifunctional RNA-binding compounds that can modulate siRNA hydrophobicity for deep penetration and gene silencing in the retina.Molecular therapy. Nucleic acids · 2025Article
- Mechanistic intracellular PK/PD modeling to inform development strategies for small interfering RNA therapeutics.Molecular therapy. Nucleic acids · 2025Article
- Differential Protein Precipitation-Based GalNAc-siRNA Sample Preparation with LC/MS Method Development Workflow in Plasma.Analytical chemistry · 2025Article
- Review
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3 authors.
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Abstract
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-quantitative 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-bp 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 RNA-induced silencing complex (RISC)-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing the
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