Technologies → qPCR

GLP/GCLP qPCR services for drug development

qPCR assay design, validation, and high-throughput analysis to meet FDA and EMA expectations for gene therapy, RNA therapeutics bioanalysis, and biomarker gene expression analysis.

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QPCR CRO

Custom qPCR assay design and validation

Absolute qPCR assays for advanced therapies

Absolute qPCR provides an exact copy number of the target nucleic acid, which is essential for regulated assays such as copy number analysis of AAVs, biodistribution, shedding, pharmacokinetics, and transgene expression in gene therapy, RNA therapeutics, mRNA vaccines, and other advanced therapies.

Absolute qPCR requires a standard curve with known concentrations of the drug product target (e.g., vector, siRNA, or other nucleic acids) or a validated substitute standard. We design, optimize, validate, and analyze absolute qPCR assays under GLP, GCLP, or ISO guidelines.

Relative qPCR assays for biomarker analysis

Relative qPCR measures fold changes in gene expression relative to an endogenous reference gene (housekeeping gene) for normalization. The qPCR results provide relative expression levels normalized against one reference gene or an average value of selected reference genes . Relative gene expression analysis is widely used in gene expression studies, drug response analysis, biomarker discovery, and differential gene expression.

We offer pre-designed panels to ensure the selection of the most stable reference gene across treatments and specialize in the design, validation, and analysis of relative gene expression qPCR assays.

Multiplex qPCR

Multiplex qPCR enables the simultaneous amplification and quantification of multiple target sequences, such as differentially expressed genes, using multiple sets of primers and probes within the same reaction. This approach increases throughput by quantifying multiple targets in a single reaction, maximizing data output from limited samples, and improving cost efficiency.

However, multiplex assay design is more complex, requiring careful optimization to minimize cross-reactivity and ensure consistent amplification efficiencies across targets. With extensive experience and advanced multichannel qPCR instruments, we specialize in designing, validating, and running high-performance multiplex qPCR assays.

Scalability and high-throughput analysis

Our scalable qPCR services range from small studies to large GCLP-compliant clinical trials. We utilize LIMS-integrated QuantStudio 12K Flex, 7 Pro, and 5 systems, supporting 96- and 384-well plates with multiplexing capabilities. The LightCycler 480 and Bio-Rad CFX Opus 96 & CFX384 Touch™ offer high-throughput, 5-plex detection for efficient assay development.

For liquid handling automation, we use QIAsymphony for high-throughput RNA/DNA extraction and the Eppendorf epMotion 5075 for automated pipetting, PCR setup, and reagent handling, ensuring precision and efficiency.

We perform qPCR on short RNA and DNA targets

qPCR assay design and validation services

Feasibility testing

Confirm amplification, specificity, and efficiency

Feasibility testing serves as a proof-of-concept to evaluate whether the qPCR assay can effectively detect and quantify the target nucleic acid under real conditions.

Primary output: Go/no-go decision for further development

Method development

Optimize reaction conditions and assay performance

Method development aims to fine-tune assay performance for optimal sensitivity, specificity, and robustness. The development includes optimizing qPCR reaction component concentrations, refining thermal cycling conditions to improve efficiency, and minimizing variability between runs.

Primary output: Optimized protocol ready for qualification

Method qualification and validation

Assay performance assessment and full regulatory-grade validation

Method qualification confirms that a qPCR method meets key criteria before full validation, including specificity, precision, LOD, LOQ, and robustness. Once qualified, regulatory-grade validation ensures compliance by assessing accuracy, linearity, dynamic range, and sample stability.

Primary output: A validated, regulatory-compliant assay for regulated applications.

Category	Feasibility	Method Development	Method Qualification	Method Validation
Validation or sample analysis study plan		Optional	Required	Required
Assay Design	3 Designs per target	3 Designs per target	3 Designs per target	3 Designs per target
Assay Efficiency and Amplicon Confirmation	√	√	√	√
Establish calibration curve parameters	√	√	√	√
Linear Range of Assay		√	√	√
Accuracy and Precision in surrogate matrix (buffer)		2 runs, 5 levels, 3 sets
Extraction Efficiency		3 kits, 1 level of spike, QC of nucleic acids Quality and Integrity, 1 repeat	1 kit, 3 levels of spike, QC of nucleic acids Quality and Integrity, 2 repeats	1 kit, 3 levels of spike, QC of nucleic acids Quality and Integrity, 2 repeats
Minimum Required Dilution (MRD)		√	√	√
Accuracy and Precision in authentic matrix		2 runs, 5 levels, 3 sets	3 runs, 5 levels, 3 repeats	6 runs, 5 levels, 3 repeats
Selectivity and Specificity in authentic matrix		Optional	√	√
Dilution linearity		Optional	√	√
Stability		Optional	Optional	√
Data QC		20%	50% or 100%	100%
QA involvement			Optional	√
Report		√	√	√
Acceptance criteria		Depending on Context of Use	Depending on Context of Use	Depending on Context of Use
QC levels		LLOQ, LQC, MQC, HQC, ULOQ	LLOQ, LQC, MQC, HQC, ULOQ	LLOQ, LQC, MQC, HQC, ULOQ

GLP for qPCR and dPCR

We are accredited for Good Laboratory Practice (GLP) by the Swedish Board for Accreditation and Conformity Assessment (SWEDAC) for qPCR, dPCR, and molecular biology.

qPCR services at TATAA

Samples

Tissues
Blood
Liquid biopsies
Fresh frozen
FFPE
EDTA
PAX

Test items

Total RNA
Messenger RNA (mRNA)
Micro RNA (miRNA)
Small interfering RNA (siRNA)
Antisense oligonucelotides (ASOs)
DNA
Single nucleotide polymorphism (SNPs)
Copy number variations (CNVs)
Cell-free DNA (cfDNA)
Minimal residual disease (MRD)
Vector copy number (VCN)
Host cell DNA residuals

What is qPCR?

qPCR, or real-time PCR, is a PCR technique in which target nucleic acid is amplified while simultaneously detecting fluorescence from either a sequence-specific fluorescent probe or an intercalating dye like SYBR Green. The fluorescence signal increases with each cycle and is measured in real-time. The PCR cycle at which the fluorescence reaches a defined threshold is called the quantification cycle (Cq).

A lower Cq value indicates that fewer cycles were needed to reach the threshold, meaning the initial DNA/RNA concentration was higher. Conversely, a higher Cq value means more cycles were required, indicating a lower initial DNA/RNA amount.

The Cq value is compared to a known standard curve in absolute qPCR to determine exact copy numbers. In relative qPCR, the Cq value is normalized against an endogenous reference gene with stable expression across all samples, tissues, and treatments to compare gene expression levels.

The qPCR assay and the qPCR method

The design and validation of a qPCR method encompass the entire qPCR workflow, including sample extraction, reverse transcription (RT) to convert RNA into cDNA, primer and probe design, amplification evaluation, and data quality control (QC) and analysis.

Each qPCR assay (primer and probe set) is specifically designed to detect and amplify the target nucleic acid with the required specificity and sensitivity for its intended use. The extraction recovery, RT, and PCR efficiency depend on multiple factors, including nucleic acid quality, sample type (tissue or biofluid), RNA degradation status, primer and probe design, and PCR reaction parameters (e.g., temperature, master composition).

Additionally, characteristics of the test item, such as whether it is free AAV DNA or encapsulated, its length, modifications, and secondary structures, can significantly influence assay performance. While experience accelerates the design process, the true performance of a qPCR method must be thoroughly evaluated and cannot be assumed.

qPCR assay controls and acceptance criteria

Each step of the method, from extraction to data analysis, requires appropriate controls. During method development, controls such as spiked-in known concentrations of test items, alien RNA, or DNA are used to monitor extraction efficiency, reverse transcription (RT) efficiency, and PCR efficiency.

A no reverse transcriptase (NTC-RT) control in cDNA synthesis should result in no amplification; otherwise, the RNA sample is contaminated with genomic DNA. Negative controls (NTC; no template controls) are essential in every PCR run to ensure that the mastermix and reagents are free from contamination.

In regulated, validated qPCR analyses, multiple QC samples with known concentrations are included in every run. These QC samples span the assay range, and all must meet the defined precision (CV%) and accuracy (RE%) criteria for the assay to pass. The calibration curve must also meet precision (CV%), accuracy (RE%), R², and efficiency criteria. All negative controls must remain negative.

Sample measurements must fall within the assay range and meet precision requirements for valid results. Reagents must be used within their validated shelf life, and equipment must be within its calibration window. Additionally, analytes and reagents must be stored within their established stability conditions to ensure assay integrity.

MIQE guidelines and regulatory requirements for qpcr

The MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) outline best practices for designing, performing, analyzing, and reporting qPCR and RT-qPCR experiments to enhance reproducibility, transparency, and reliability in qPCR-based studies.

TATAA Biocenter co-authored the MIQE guidelines, and these principles are deeply embedded in our workflows, ensuring rigorous use of controls and standardized processes.

Regulatory guidance from the FDA and EMA on qPCR assay validation remains incomplete and inconsistent. However, in 2024, TATAA Biocenter, in collaboration with 37 industry experts from 24 organizations, published the paper ‘Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development’ in AAPS, providing industry-aligned best practices.