bioanalysis
CASE STUDY
Background
Oligonucleotide therapeutics are advancing rapidly, with an increase in demand of analysing the oligos for pharmacokinetics and biodistribution studies. Traditional platforms, including LC-MS and ligand-binding assays, often struggle to deliver the sensitivity and sequence specificity needed.
Quantitative PCR addresses that sensitivity gap, but conventional qPCR workflows carry their own burden: labor-intensive nucleic acid extraction, matrix-dependent inhibition, and the need for matrix-matched standard curves at every step. The cumulative effect is longer timelines, higher costs, and more variables to manage during method development.
The Challenge
For sponsors quantifying oligonucleotides across plasma and multiple tissue types, the standard approach creates real friction. Extraction steps consume time and samples. Matrix-driven Ct shifts require extensive optimization work. And running separate standard curves for each tissue type adds complexity at every stage of a study.
The question was whether it was possible to eliminate extraction entirely while maintaining the analytical performance required for regulated bioanalysis, across all relevant matrices, from a single calibration platform.
Our Approach
Resolian’s team developed and systematically evaluated an extraction-free, direct-to-qPCR workflow using optimized detergent-based homogenization combined with controlled matrix dilution. Plasma and non-human primate tissues were lysed using detergents and proteinase K. Rather than extracting nucleic acids, samples were serially diluted to a point where matrix inhibition was effectively neutralized while maintaining the signal to noise ratio.
The critical finding came from evaluating dilution ranges across seven biological matrices. At 600x dilution with a harsh detergent buffer, standard curves for plasma and all tissue types converged to near-identical slopes, intercepts, and PCR efficiencies, with R-squared values above 0.998 across all matrices. This meant a single plasma-based standard curve could accurately quantify oligonucleotides across diverse tissues without meaningful loss of accuracy.
Assay performance was confirmed across a broad dynamic range (100 to 10 to the eighth copies per microlitre), using a 75-nucleotide single-stranded DNA target as a model analyte, with QC recoveries consistently falling within the 80-120% acceptance window.
Running Oligonucleotide Studies Across Multiple Matrices?
Resolian’s team has developed and validated extraction-free qPCR workflows for oligonucleotide quantitation across plasma and tissue types, cutting method development time without compromising performance.
Results
The unified workflow delivered several concrete outcomes:
- A single common standard curve supports both plasma and six NHP tissue types at 600x dilution
- All matrices showed excellent linearity (R-squared above 0.998) and PCR efficiencies near 100%
- HQC and LQC recoveries were consistent across matrices, with minimal inter-matrix bias
- Hands-on time and per-sample cost were substantially reduced significantly by removing the extraction step
- Run-to-run variability was minimized through uniform baseline correction and ROX normalisation
What This Means
This work demonstrates that extraction-free qPCR is not a compromise: it is a viable, high-performance approach for multi-matrix oligonucleotide bioanalysis. For sponsors running tissue distribution studies or PK/PD programmes across multiple species, a unified platform means fewer resources spent on method development and more confidence in the data. Resolian is actively expanding this framework to additional tissue types and adapting the approach for one-step RT-qPCR to support RNA quantification.
Resolian partners with pharma and biotech companies to solve complex bioanalytical challenges, enabling faster, more reliable decisions in drug development.
Ready to Simplify Your Oligonucleotide Bioanalysis?
Resolian partners with pharma and biotech companies to deliver high-throughput qPCR and oligonucleotide bioanalytical solutions, from method development through multi-matrix study support.
