An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1

Alison S. Devonshire, Rebecca Sanders, Alexandra S. Whale, Gavin J. Nixon, Simon Cowen, Stephen L.R. Ellison, Helen Parkes, P. Scott Pine, Marc Salit, Jennifer McDaniel, Sarah Munro, Steve Lund, Satoko Matsukura, Yuji Sekiguchi, Mamoru Kawaharasaki, José Mauro Granjeiro, Priscila Falagan-Lotsch, Antonio Marcos Saraiva, Paulo Couto, Inchul YangHyerim Kwon, Sang Ryoul Park, Tina Demšar, Jana Žel, Andrej Blejec, Mojca Milavec, Lianhua Dong, Ling Zhang, Zhiwei Sui, Jing Wang, Duangkamol Viroonudomphol, Chaiwat Prawettongsopon, Lina Partis, Anna Baoutina, Kerry Emslie, Akiko Takatsu, Sema Akyurek, Muslum Akgoz, Maxim Vonsky, L. A. Konopelko, Edna Matus Cundapi, Melina Pérez Urquiza, Jim F. Huggett, Carole A. Foy

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)15-28
Number of pages14
JournalBiomolecular Detection and Quantification
Volume8
DOIs
StatePublished - Jun 1 2016
Externally publishedYes

Keywords

  • Biomarker identification and validation
  • Cancer
  • Diagnostics
  • Gene expression
  • Molecular diagnostic
  • Normalisation
  • RNA copy number ratio
  • RT-qPCR
  • Standardisation
  • Transcriptomics

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