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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.

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In general, research experimentation is often used mainly when new methodologies are being developed or existing ones are being improved. The characteristics of any method depend on its factors or components. The planning techniques and analysis of experiments are basically used to improve the analytical conditions of methods, to reduce experimental labour with the minimum of tests and to optimize the use of resources (reagents, time of analysis, availability of the equipment, operator time, etc). These techniques are applied by the identification of variables (control factors) of a process that have the most influence on the response of the parameters of interest, by attributing values to the influential variables of the process in order that the variability of response can be minimum, or the obtained value (quality parameter) be very close to the nominal value, and by attributing values to the influential variables of the process so that the effects of uncontrollable variables can be reduced. In this central composite design (CCD), four permanent modifiers (Pd, Ir, W and Rh) and one combined permanent modifier W + Ir were studied. The study selected two factors: pyrolysis and atomization temperatures at five different levels for all the possible combinations. The pyrolysis temperatures with different permanent modifiers varied from 600 ˚C to 1600 ˚C with hold times of 25 s, while atomization temperatures ranged between 1900 ˚C and 2280 ˚C. The characteristic masses for As were in the range of 31 pg to 81 pg. Assuming the best conditions obtained on CCD, it was possible to estimate the measurement uncertainty of As determination in water samples. The results showed that considering the main uncertainty sources such as the repetitivity of measurement inherent in the equipment, the calibration curve which evaluates the adjustment of the mathematical model to the results and the calibration standards concentrations, the values obtained were similar to international interlaboratorial comparison results.

pH is an important parameter to control the production of food