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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.

This chapter presents and explains the most used methodologies for the evaluation of measurement uncertainty in metrology with practical examples. The main topics are basic concepts and importance, existing documentation, the harmonized methodology of the Guide to the Expression of Uncertainty in Measurement, types of uncertainty, modeling of measurement systems, use of alternative methods (including the GUM supplement 1 Monte Carlo numerical method), evaluation of uncertainty for calibration curves, correlated uncertainties, uncertainties arising from the calibration of instruments, and the main proposals for the new revised GUM. The chapter also discusses the GUM as a tool for the technical management of measurement processes.

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pH is a widely used control parameter for several industrial processes. Thus, its correct determination and uncertainty estimation are extremely important. The Guide to the Expression of Uncertainty in Measurement (ISO-GUM) has been extensively used for pH uncertainty estimation. This work uses Monte Carlo simulation to estimate pH uncertainty in a primary pH system for the measurements of a regional comparison (SIM 8.11P-1) in which INMETRO has participated. The results are compared with the ISO-GUM analytical estimation approach and good agreement was found.