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.
OBJECTIVE
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.
Span
P N
PN
Department of Chemical Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. p.span@ace.umcn.nl
Sieuwerts
A M
AM
Heuvel
J J T M
JJ
Spyratos
F
F
Duffy
M J
MJ
Eppenberger-Castori
S
S
Vacher
S
S
O'Brien
K
K
McKiernan
E
E
Pierce
A
A
Vuaroqueaux
V
V
Foekens
J A
JA
Sweep
F C G J
FC
Martens
J W M
JW
D001943:Breast Neoplasms / Q000235:genetics*; D002138:Calibration; D045744:Cell Line, Tumor; D005060:Europe; D005260:Female; D005819:Genetic Markers; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007753:Laboratories / Q000592:standards*; D010338:Pathology, Clinical; D011379:Prognosis; D020033:Protein Isoforms; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D011795:Surveys and Questionnaires; D014157:Transcription Factors / Q000235:genetics; D000097577:Homeobox Protein PITX2
D005819:Genetic Markers; D018398:Homeodomain Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2