Harmonisation of multi-centre real-time reverse-transcribed PCR results of a candidate prognostic marker in breast cancer: an EU-FP6 supported study of members of the EORTC - PathoBiology Group

Eur J Cancer. 2009 Jan;45(1):74-81. doi: 10.1016/j.ejca.2008.09.030. Epub 2008 Nov 12.

Abstract

Aim: Assessment of intra- and inter-laboratory variation in multi-centre real-time reverse-transcribed PCR (qRT-PCR)-based mRNA quantification of a prognostic marker in breast cancer using external quality assurance (EQA).

Methods: A questionnaire on the methodologies used and EQA calibrators were sent to 5 participating laboratories from 4 European countries, which measured mRNA levels of PITX2 splice variants and reference genes by qRT-PCR.

Results: Differences in the methodology included PCR quantification methodology and equipment, RNA extraction and cDNA synthesis procedures. The intra-laboratory coefficient of variation (CV) ranged from 5 to 23%, and the inter-laboratory CV ranged from 17 to 30%. The inter-laboratory CV was reduced to 13% by using prediluted calibrators and by harmonising the data in the central QA laboratory. Additional normalisation using reference genes did not decrease the variation further.

Conclusions: Both externally provided calibrators and centralised harmonisation are required to reduce the intra-laboratory variation in multi-centre qRT-PCR results to an acceptable level.

Publication types

  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / genetics*
  • Calibration
  • Cell Line, Tumor
  • Europe
  • Female
  • Genetic Markers
  • Homeobox Protein PITX2
  • Homeodomain Proteins / genetics
  • Humans
  • Laboratories / standards*
  • Pathology, Clinical*
  • Prognosis
  • Protein Isoforms
  • Quality Control*
  • RNA, Messenger / analysis
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards*
  • Surveys and Questionnaires
  • Transcription Factors / genetics

Substances

  • Genetic Markers
  • Homeodomain Proteins
  • Protein Isoforms
  • RNA, Messenger
  • Transcription Factors