Ramsden Simon C, Daly Sarah, Geilenkeuser Wolf-Jochen, Duncan Graeme, Hermitte Fabienne, Marubini Ettore, Neumaier Michael, Orlando Claudio, Palicka Vladimir, Paradiso Angelo, Pazzagli Mario, Pizzamiglio Sara, Verderio Paolo
National Genetics Reference Laboratory (Manchester), St. Mary's Hospital, Manchester, UK, and Institute of Medical Statistics and Biometry, University of Milan, Italy.
Clin Chem. 2006 Aug;52(8):1584-91. doi: 10.1373/clinchem.2005.066019. Epub 2006 Jun 1.
Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR.
The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number.
The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants.
This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.
