title: An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.
abstract: Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: LGC, Teddington, United Kingdom.
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authors.affiliation: National Institute of Standards and Technology (NIST), Gaithersburg, USA.
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authors.affiliation: National Institute of Standards and Technology (NIST), Gaithersburg, USA.
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authors.affiliation: National Institute of Standards and Technology (NIST), Gaithersburg, USA.
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authors.affiliation: National Institute of Standards and Technology (NIST), Gaithersburg, USA.
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authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
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authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
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authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan; National Metrology Institute of Japan (NMIJ), Ibaraki, Japan.
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authors.affiliation: National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, Brazil.
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authors.affiliation: National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, Brazil.
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authors.affiliation: National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, Brazil.
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authors.affiliation: National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, Brazil.
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authors.affiliation: Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea.
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authors.affiliation: National Institute of Metrology Thailand (NIMT), Pathumthani, 12120, Thailand.
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authors.affiliation: National Measurement Institute Australia (NMIA), Lindfield, Australia.
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authors.affiliation: National Measurement Institute Australia (NMIA), Lindfield, Australia.
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authors.affiliation: National Measurement Institute Australia (NMIA), Lindfield, Australia.
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authors.forename: Akiko
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authors.affiliation: TÜBİTAK Ulusal Metroloji Enstitüsü (UME), Kocaeli, Turkey.
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authors.lastname: Akgoz
authors.affiliation: D.I. Mendeleev Institute for Metrology (VNIIM), St. Petersburg, Russian Federation; Institute of Cytology RAS, St. Petersburg, Russian Federation.
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authors.affiliation: D.I. Mendeleev Institute for Metrology (VNIIM), St. Petersburg, Russian Federation.
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authors.affiliation: Centro Nacional de Metrologia (CENAM), Querétaro, Mexico.
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authors.affiliation: Centro Nacional de Metrologia (CENAM), Querétaro, Mexico.
authors.forename: Melina Pérez
authors.initials: MP
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authors.affiliation: LGC, Teddington, United Kingdom.
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keywords: Biomarker identification and validation; Cancer; Diagnostics; Gene expression; Molecular diagnostic; Normalisation; RNA copy number ratio; RT-qPCR; Standardisation; Transcriptomics