Quantitative PCR (qPCR), digital PCR (dPCR), and Next-Generation Sequencing (NGS) are essential technologies in siRNA bioanalysis. They provide the precise quantification, sensitivity, and specificity needed to track siRNA’s therapeutic efficacy, stability, and biodistribution.
Background
Small interfering RNA (siRNA) is a component of RNA interference (RNAi), a natural regulatory process that silences target mRNA in a sequence-specific manner. In therapeutic applications, siRNA suppresses gene expression by induce degradation of target mRNA, offering the potential for treating conditions like genetic disorders and cancers. Delivery methods for siRNA include nanoparticle-based systems, such as lipid nanoparticles (LNPs), polymer-based carriers, and viral vectors. Delivery challenges include ensuring siRNA stability and cellular uptake and minimizing off-target effects. Chemical modifications enhance the stability and half-life of siRNA, making it more effective in therapeutic applications. GalNAc-siRNA conjugates improve targeting to specific tissues, such as the liver. Bioanalysis of siRNA involves monitoring stability, cellular uptake, and gene-silencing efficacy, often using matrices such as blood, plasma, and tissues. Critical assessments include pharmacokinetics (PK) to track biodistribution and pharmacodynamics (PD) to measure gene-silencing and gene knockdown effects.
Monitoring the mechanism
Gene silencing with siRNA begins when double-stranded RNA (dsRNA) is processed by the Dicer enzyme, which cleaves it into siRNA fragments. These fragments are then loaded into the RNA-induced silencing complex (RISC), where they guide the complex to bind to and degrade target mRNA, effectively silencing gene expression.
Using custom, validated real time PCR (RT-PCR) assays, quantitative PCR (qPCR) and digital PCR (dPCR) quantify the reduction in target mRNA levels, providing precise measurements of gene-silencing and gene knockdown efficiency and its kinetics over time. With our Two-Tailed PCR technology (described below), we can quantify short targets, such as siRNA, with validated qPCR and dPCR assays to track their abundance across different tissues and their stability and persistence. qPCR and dPCR can also measure Dicer activity by quantifying pre-siRNA and mature siRNA, assess AGO2 activity by measuring cleaved mRNA fragments, and quantify the guide strand associated with RISC after a RISC pulldown.
NGS enables a comprehensive gene expression analysis, detecting and validating global changes and potential off-target effects. It also allows pathway enrichment analysis to identify broader gene-silencing impact on cellular processes.
Primer design using TATAA’s Two Tailed PCR Technology
Primer design for siRNA quantification is challenging due to the short length of siRNA sequences. A common approach is to elongate the target with a stem-loop primer; however, this method offers limited flexibility, restricting options for optimization. PCR optimization is often necessary to meet the performance criteria required for a validated assay. TATAA Biocenter’s Two Tailed PCR technology addresses these challenges by using dual-binding hemiprobes that bind the siRNA, enhancing specificity compared to traditional stem-loop primers that bind only one end. Additionally, the hemiprobes allow flexibility in positioning the primer binding sites along the siRNA, optimizing the PCR reaction. Two Tailed PCR enables flexibility in primer design, optimizing for various RT-PCR conditions while maintaining sensitivity and reducing non-specific amplification. This technology can be validated to the same standards as other PCR assays, ensuring reliable results and enhancing the robustness of siRNA bioanalysis.
Method development
A custom assay is required because the target sequence varies between siRNA constructs and mRNA silencing targets, necessitating target-specific assays for accurate detection and quantification. Assay performance is affected by primer design, modifications of the siRNA, secondary structures, and the sample matrix. Method development focuses on optimizing PCR conditions to achieve high sensitivity, specificity, and selectivity for the target. This process includes refining extraction protocols to determine recovery yield and extraction efficiency and adjusting primers and probes. Each parameter is rigorously tested during method development to ensure assay efficiency before moving on to validation.
Method validation
Method validation is conducted using blank samples spiked with known target concentrations. Key assay parameters, including limit of detection (LOD), lower limit of quantification (LLOQ), and upper limit of quantification (ULOQ), are assessed for precision and accuracy to ensure minimal variability and close alignment with the true value across multiple runs, analysts, and days. Validation also involves evaluating extraction performance, sample stability, and PCR efficiency across a concentration range. The depth of validation is tailored to the assay’s intended use, whether for research purposes or regulatory compliance.
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