The Nuclease Protection Assay (NPA) detects, quantifies, and maps specific RNA via labeled complementary probes. It uses single-strand-specific nucleases to degrade non-target material, ensuring high specificity and low background, ideal for critical RNA analysis. Creative Biolabs' NPA service offers ultra-sensitive solution-phase hybridization, aiding accurate quantification of low-abundance RNA, distinguishing related transcripts, and validating mRNA integrity to accelerate drug development and boost regulatory confidence.
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Developed decades ago, the Nuclease Protection Assay (encompassing both RNase Protection Assay and S1 Nuclease Assay) remains a crucial technique because its specificity is based on the biochemical principle of perfect hybridization, which is then stringently validated by enzymatic exclusion. Unlike techniques reliant on amplification or real-time fluorescence, NPA provides a direct measure of the intact target, making it the ultimate tool for confirming structural integrity and accurate quantification, especially for therapeutic oligonucleotides and mRNA constructs.
Creative Biolabs offers both traditional and modernized NPA methods tailored to client needs:
The Creative Biolabs NPA workflow is meticulously designed for high-quality, reproducible results and is frequently adapted for high-throughput analysis, such as integration with the Meso Scale Discovery (MSD) platform for enhanced speed.
| Stage | Activity Description |
|---|---|
| Required Starting Materials | High-quality total RNA samples (from cells, tissues, or in vivo studies), target gene sequence information (mRNA or oligo drug), and detailed sample matrix descriptions (e.g., plasma, liver homogenate, CSF). |
| Probe Design and Synthesis | Design and synthesize a radiolabeled or electrochemiluminescence-labeled complementary oligonucleotide or RNA probe specific to the target sequence, ensuring it spans the region of interest for maximum specificity. |
| Hybridization | The labeled probe is mixed with the client's RNA sample. The solution is incubated under optimized conditions (time, temperature) to allow the probe to anneal specifically to the target RNA molecule. |
| Nuclease Digestion (The Double Filter) | A single-strand-specific nuclease (e.g., S1 Nuclease or RNase cocktail) is added to the solution to catalytically degrade all single-stranded nucleic acids (unhybridized probe and non-target RNA). |
| Analysis and Quantification | The protected fragments are separated by high-resolution gel electrophoresis (for mapping) or quantified using the MSD electrochemiluminescence platform (for PK studies). |
Final Deliverables:
The typical timeframe for this service ranges from 4 to 8 weeks, depending on the complexity of the target sequence and the extent of assay validation required for regulatory submissions.
Our Nuclease Protection Assay Service at Creative Biolabs is designed to eliminate the ambiguity and matrix interference often encountered with standard RNA quantification methods. We don't just run assays; we provide custom-optimized analytical solutions that deliver regulatory-grade data for your most critical nucleic acid projects.
Customized Assay Development (The NPA Gold Standard)
We offer fully tailored Nuclease Protection Assay protocols, including both RNase Protection Assay (RPA) and S1 Nuclease Assay, optimized for your specific RNA or oligonucleotide drug target and biological matrix.
Ultra-Sensitive PK/PD Quantification
We provide custom NPA-MSD integration for high-throughput, ultra-sensitive (pg/mL), and large dynamic range quantification of therapeutic oligonucleotides (ASOs, siRNAs) in challenging matrices like plasma, CSF, or tissue homogenates.
High-Resolution Structural Validation
Precise mapping of transcription starts and termination sites and intron/exon boundaries for validating the structural integrity and quality control of IVT mRNA constructs.
Unrivaled Specificity for Complex Samples
The unique nuclease cleanup step is rigorously customized to minimize background noise and eliminate matrix effects, guaranteeing unambiguous quantification that easily distinguishes between highly homologous transcripts (e.g., multigene families).
Support for Regulatory Milestones
Our data package, including assay validation metrics (sensitivity, dynamic range, reproducibility), is designed to support critical preclinical stages and accelerate IND-enabling studies.
Expert Consultation and Optimization
Direct access to our 20+ years of molecular biology expertise for sequence-based probe design and full workflow optimization to maximize yield and confidence in your results.
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While qPCR is rapid, NPA offers superior confidence. NPA's nuclease digestion step ensures that only perfectly hybridized targets are measured, eliminating concerns about non-specific amplification or primer-dimers often seen in qPCR. This makes NPA the "Gold Standard" for quantifying critical, low-abundance therapeutic molecules where absolute specificity is non-negotiable.
Absolutely. NPA is highly tolerant of complex biological matrices like tissue homogenates. The initial hybridization step is followed by the powerful nuclease cleanup, effectively dissolving matrix interference and ensuring accurate quantification of your oligonucleotide drug, even in low-volume, complex samples.
While traditionally a more laborious method, Creative Biolabs has successfully adapted the NPA workflow for high-throughput applications by integrating it with the Meso Scale Discovery (MSD) electrochemiluminescence platform. This allows for the rapid processing and quantification of a large number of samples, making it suitable for preclinical PK/PD screening.
To ensure a rapid start, we primarily need the sequence of your target RNA or oligonucleotide drug, the type of biological matrix (e.g., plasma, liver, CSF), and the expected concentration range. This information allows us to immediately initiate specialized probe design and assay optimization tailored to your specific project needs.
Creative Biolabs is the trusted partner for your most demanding nucleic acid analysis projects. Our Nuclease Protection Assay Service delivers unambiguous, sensitive, and high-resolution data essential for validating construct integrity and advancing therapeutic candidates through preclinical development. We turn complex analytical challenges into clear, actionable results.
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