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EQUAL-quant: an international external quality assessment scheme for real-time PCR
Simon C Ramsden1, Sarah Daly, Wolf-Jochen Geilenkeuser
1National Genetics Reference Laboratory (Manchester), St. Mary's Hospital, Manchester, UK, and Institute of Medical Statistics and Biometry, University of Milan, Italy. simon.ramsden@cmmc.nhs.uk
Clinical Chemistry
|June 3, 2006
Summary
External quality assessment (EQA) for real-time PCR using TaqMan probes revealed significant performance variations among 137 laboratories. This highlights the need for standardized methods in quantitative gene expression analysis for diagnostics.
Area of Science:
- Molecular Biology
- Diagnostic Assays
- Quality Assurance
Background:
- Quantitative gene expression analysis via real-time PCR is crucial for leukemia MRD detection and cancer prognosis.
- The European Union's EQUAL project aims to establish external quality assessment (EQA) for diagnostic and research labs.
- The EQUAL-quant program specifically evaluates TaqMan probe assays, a common real-time PCR method.
Purpose of the Study:
- To assess the technical performance and quality standards of real-time PCR TaqMan assays across European laboratories.
- To identify variability in RNA extraction, reverse transcription, and DNA quantification.
- To establish the need for external quality assessment in this field.
Main Methods:
- Development of the EQUAL-quant reagent set for standardized assay evaluation.
- Implementation of a multidisciplinary EQA scheme involving 137 laboratories from 29 countries.
- Statistical analysis of results to assess precision and accuracy of reported data.
Main Results:
- Significant performance differences were observed among participating laboratories.
- Twenty percent of laboratories failed to meet precision and/or accuracy criteria.
- No performance variations were linked to the diverse testing platforms (>10) utilized.
Conclusions:
- The EQA scheme confirms a strong need and demand for external assessment of real-time PCR technical standards.
- The developed reagents and statistical tools can serve as a benchmark for acceptable working standards.
- This initiative supports the advancement of reliable quantitative gene expression analysis in diagnostics.
