Nucleic acid quantification--progress and pitfalls.

Abstract

Half a century after the Nobel Prize for Physiology or Medicine was jointly attributed to Francis Crick, James D. Watson, and Maurice Wilkins “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material,” the development and use of accurate methods to quantify nucleic acids in their different forms still remain a challenging exercise. Toomany “scientific” results using inappropriate or incomplete analytical procedures have been published. A “quick and dirty” estimation of the total nucleic acid concentration followed by an uncontrolled extraction step generating a solution enriched in nucleic acid that is partially amplified during a polymerase chain reaction (PCR) or hybridized on a microarray is often providing results that cannot be repeated or are strongly biased. Despite the increasing use of measurement results for nucleic acid amounts in decision making, may it be in the frame of genetically modified organisms (GMO) trade or in medical diagnostics, until recently more efforts have been directed (e.g., by instrument developers) to increase the sample throughput rather than to improve the quality of analytical data. The lack of reproducibility and comparability of measurement results as well as of standardized procedures represent major hurdles for translating experimental findings into sound generic knowledge and new tools, required for instance in healthcare. Two kinds of actions seem to be necessary for improving the balance between quantity (amount) and quality of data: on the one hand raising awareness about existing ‘good practice’ guidelines and stimulating their application and on the other hand developing and disseminating new approaches for modern analytical quality assurance and metrology. Both activities should be supported by scientific publishing. Therefore, this topical collection of Analytical Bioanalytical Chemistry is devoted to problems related to the quantification of different nucleic acid molecules. In the following, mRNA used for gene expression measurement by reverse transcription quantification PCR (Sanders et al.), standardization of measuring cell free DNA in plasma (Devonshire et al.), and effects of methylated CpG sites (Burke et al.) are dealt with. Efforts to obtain reliable quantification of GMO (Milavec et al.) and pathogenic bacteria in plants (Dreo et al.) are also discussed. From a conceptual point of view, nucleic acid quantification is not different from any other chemical measurement. It is indeed a multistep process, starting from adequate sampling, followed by an extraction procedure, quantification itself, and finishing with data analysis and reporting of the results, including an appropriate measurement unit. Uncertainty components are associated with all those steps and they contribute to the variability of the final result. Sanders et al. have reviewed the different parameters that affect the quantification of mRNA by reverse transcription quantitative PCR. The importance of appropriate RNA isolation and storage conditions, and of the choice of relevant reference genes are discussed, as well as the difficulty in coping with the inherent biological variability of patients. It is also stressed that the upstream process, prior amplification by the polymerase, is creating the major contributions to the variability of results. The importance of an appropriate extraction protocol during the quantification of nucleic acids in “liquid biopsy” is Published in the topical collectionNucleic Acid Quantificationwith guest editors Hendrik Emons and Philippe Corbisier. H. Emons (*) : P. Corbisier Institute for Reference Materials and Measurements (IRMM), Joint Research Centre, European Commission, Retieseweg 111, 2440 Geel, Belgium e-mail: hendrik.emons@ec.europa.eu