qPCR and dPCR assay design and validation services

When a qPCR, dPCR, or other molecular assay has been validated for regulatory purposes in one laboratory and needs to be transferred to another, partial validation is typically required. This ensures the assay performs equivalently under the new laboratory’s conditions. The receiving lab must demonstrate comparable performance across critical parameters such as accuracy, precision, sensitivity (LOD/LOQ), linearity, specificity, and robustness.

At TATAA Biocenter, we support method transfer, laboratory transfer, and analytical transfer by offering a broad range of instrumentation that matches or complements your existing setup. We perform cross-validation studies to confirm assay equivalence, enabling a seamless transition and uninterrupted sample analysis for ongoing regulated studies or new clinical phases.

The standard rule is to position the primers over a non-natural junction to prevent nonspecific binding of endogenous DNA or transcribed RNA. Suppose the introduced gene is identical to the endogenous gene. In that case, the optimal primer location is at the promoter-target junction, the junction between the gene and the post-translational enhancement element, or over an exon-exon junction to ensure that only the vector DNA is detected.

If the assay should only detect expressed transcripts, it is crucial to design it to avoid detecting endogenous transcripts. To achieve specificity, primers cover vector-specific elements that are also transcribed, such as promoters, enhancers, or regulatory elements.

If one cannot distinguish the vector-derived transcript from the vector DNA, then one must perform DNA removal before conducting RT-PCR. Additionally, including a No Reverse Transcriptase (RT-negative control) becomes necessary to account for copies of contaminating vector DNA. If the RT-negative control, the RT-PCR without enzyme detects any signal it is originating from vector DNA sequences present before the RT-PCR. When the introduced gene is codon-optimized and has a different sequence than the endogenous gene, primer and probe sequences can target regions with the least similarity to the endogenous sequence.

The primers or probe should focus on the unnatural exon-exon junction in exon-skipping therapies.

Designing primers and probes to target the vector backbone independently of the delivered transgene in early development reduces assay development efforts by employing a universal PCR assay.

TATAA Biocenter applies the highest qPCR validation guidelines and dPCR primer design standards, ensuring accuracy and reproducibility in biomarker analysis and gene quantification.

The MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) define best practices for designing, optimizing, and reporting qPCR and RT-qPCR experiments. These guidelines enhance the reproducibility, transparency, and reliability of qPCR-based studies.

As a co-author of the MIQE guidelines, TATAA Biocenter integrates these principles into every stage of assay development, using stringent controls and standardized workflows to meet the highest quality standards.

Despite the absence of fully harmonized qPCR validation guidelines from regulatory agencies such as the FDA and EMA, TATAA Biocenter has been leading in defining industry best practices. In 2024, with 37 experts from 24 organizations, we co-authored the paper “Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development” in AAPS. This publication provides a unified framework for qPCR validation and dPCR assay optimization, including guidance on dPCR primer design and key considerations for assay robustness.

With a thoroughly developed method, the validation process becomes straightforward. The time-consuming aspect is the method development, which can be significantly expedited by leveraging expert knowledge and experience. By using established workflows, best practices for design, and carefully selecting kits and instruments, the optimization time can be greatly reduced.

Method development encompasses every step, from the raw organ specimen to the final result.

Biopsy sampling: Considerations on how the dissection is performed, what surrounding tissue may be included, the composition of cell types or organ pieces, whether the sample should be subdivided into different parts of the organ or tumor, and how it is frozen. The type of anticoagulant and tube used are essential for biofluids to simplify downstream extraction.

Tissue processing: Homogenization or lysis, depending on the specific tissue type.

Nucleic acid extraction: This process depends on whether RNA or DNA is being extracted, any modifications, the length of the nucleic acids, and other relevant features. Ideally, the extraction recovery should be checked with the actual test item, as control spikes may show more uniform performance across different kits.

Automation: The extraction process is automated using one of our liquid handlers for standardization. We have established workflows that yield high-quality RNA and DNA from common tissues and biofluids, such as RNA extraction from blood using PAX tubes. We also perform manual extractions when it is the best option for the sample.

cDNA synthesis: Not all RT enzymes perform equally well with the same target and matrix. Target availability is also crucial, as secondary structures may interfere with primer binding. The choice of cDNA primer, whether gene-specific priming, oligo(dT) primers, or random hexamers, can significantly influence cDNA synthesis and, consequently, the quantification accuracy.

Primer and probe design: Optimized to enhance PCR efficiency, specificity, and selectivity, with strategies employed to discriminate between similar sequences.

Data normalization: The samples are normalized against mass/volume, cell number, total RNA/DNA amount, alien spike, or ribosomal RNA, depending on the assay circumstances. When using a reference gene, we evaluate and select the one that demonstrates the most stability across experimental conditions and has an expression level similar to the target gene.

Quality control (QC): All non-automated steps are QC-tested, including data transfer steps and calculations.

The analytical performance characteristics are determined during method development and later confirmed during validation. These characteristics include primer and probe set evaluation, assessing qPCR efficiency, and positive/negative separation in digital PCR (dPCR). Additionally, PCR optimization, calibration curve optimization (for qPCR), extraction optimization, recovery, extraction efficiency, precision, accuracy, specificity, selectivity, and sensitivity of the assay are thoroughly evaluated.

Assay validation involves validating the entire process, from tissue sample preparation to target concentration measurement.

The performance characteristics tested during method validation include the assay’s precision, accuracy, PCR efficiency, dilutional linearity, and co-linearity. The sensitivity of the assay is confirmed by determining the limit of blank (LOB), the limit of detection (LOD), and the lower limit of quantification (LLOQ). Specificity and selectivity are tested using matrices without the test items. Sample stability and extraction efficiency are also assessed. The robustness and ruggedness of the assay are evaluated by running the assay multiple times, with different analysts, on separate days and using different instruments.

The validation is performed on spiked blank samples, also used as assay performance controls during sample analysis (QC samples) and for the calibration curve (qPCR only). A matrix is a sample devoid of the analyte and should ideally match the biological sample. The reference material should, if possible, be the drug product. Still, it could also be plasmid DNA for biodistribution studies, whether the DNA is free in the cytosol or encapsulated DNA for shedding assays to ensure that DNA is released during extraction. QC samples and calibrators should be derived from different stock solutions.

The intra- and inter-assay precision should be ≤ 30% coefficient of variation (%CV) for QCs and ≤ 50% CV for LOQs, whether using interpolated qPCR results or absolute dPCR copy number results.

Intra- and inter-assay accuracy for qPCR should range from –50% to +100% relative error (RE) on interpolated copies. This is because the doubling nature of qPCR means that a difference of just 1 Cq can result in the interpolated result being either half or twice the nominal concentration. For dPCR, the inter-assay accuracy for absolute copy numbers should have a relative error (%RE) of ≤ 30% for QCs and ≤ 50% for LOQs.

A PCR efficiency of 100% means that all target sequences are doubled in each cycle. PCR efficiencies between 90% and 110% are acceptable, as demonstrated by a slope between −3.1 and −3.6 in the standard curve. The linear regression should have an R² value of ≥ 0.98.

Dilution linearity demonstrates that the slope remains linear even when the sample is diluted. Co-linearity refers to the scenario where multiple matrices show similar linearity within the assay range, allowing one matrix to be used as a surrogate for the others.

The limit of blank (LOB) is determined as the concentration at which the sample is negative with 95% confidence, while the limit of detection (LOD) is the concentration at which the sample is positive with 95% confidence.

Specificity and selectivity are confirmed when 100% of the unspiked matrices show results below the LOD, and at least 8 out of 10 spiked samples meet the precision and accuracy criteria for the LLOQ.

Stability is confirmed using QC samples that undergo the exact handling as the actual samples, including long-term stability testing. The extraction efficiency is validated by spiking the sample and then measuring the extraction efficiency and recovery.

A sample analysis run includes QC samples prepared in sets for each plate at different concentration levels, calibrators for qPCR, and blank matrices as negative controls. RT-NTC (Reverse Transcription No-Template Control) can be included to check for DNA contamination in the cDNA.

The run is accepted if all the QC samples and the calibration curve meet the defined precision and accuracy criteria. The R² value of the calibration curve must also meet the required threshold. Additionally, all negative controls must be negative, and the analyzed sample must fall within the assay’s range.