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

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Research Article: An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1
ผู้เขียน/Author: Alison S.Devonshirea, Rebecca Sanders, Alexandra S.Whale, Gavin J. Nixon, Simon Cowen, Stephen L.R. Ellison, Helen Parkes, P. Scott Pineb, Marc Salit, Jennifer McDaniel, Sarah Munrob, Steve Lund, Satoko Matsukura, Yuji Sekiguchic, Mamoru Kawaharasaki, José Mauro Granjeiro, Priscila Falagan-Lotsch, Antonio Marcos Saraiva, Paulo Couto, Inchul Yang, Hyerim Kwon, Sang-Ryoul Park, Tina Demsar, Jana Zel, 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
Email: duangkamol.vir@siam.edu
Department/Faculty Faculty of Nursing,  Siam University, Bangkok 10160
Published/แหล่งเผยแพร่ Biomolecular Detection and Quantification Volume 8, June 2016, Pages 15-28

Citation

Devonshirea A. S., Sanders R., Whale A. S., Nixon G. J., Cowen S., Ellison S. L.R.,… Foy, Carole A. (2016). An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1. Biomolecular Detection and Quantification, 8(2016), 15-28.


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.

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


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

Faculty of Nursing, Siam University, Bangkok, Thailand

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