Improving the analysis of quantitative PCR data in veterinary research

Abstract

! 2011 Elsevier Ltd. All rights reserved. In recent years we have seen the emergence of molecular veterinary medicine, prompted by the availability of sequence information on pathogens, as well as on mammalian species relevant for economic (e.g. chicken, pig, cattle) and social (e.g. horse, dog, cat) purposes. Molecular biological tools are being integrated rapidly in the diverse specialities of veterinary research, allowing diseases to be described in molecular terms, both at the genetic level (DNA) and at the functional genomics level, including mRNA, non-coding RNA (ncRNA) and microRNA. Species-specific single nucleotide polymorphism (SNP) platforms are available to resolve the genetic background of inherited diseases. Expression profiling or functional genomics aims to identify differential gene signatures or ncRNA profiles associated with various experimental and/or clinical conditions. This is commonly carried out using species-specific micro-array platforms that measure relative expression levels of large numbers of gene products or miRNAs. Results need to be verified using independent techniques; the most popular validation technique is real-time, fluorescence based, reverse transcription quantitative PCR (RT-qPCR). The highly sensitive RT-qPCR technique is a complex, multi-step procedure that can be prone to numerous errors, potentially leading to misinterpretation of data. Hence, it is important that the technical information provided in a manuscript is complete, that protocols are validated and that results and conclusions are based on appropriate methods of analysis. Numerous publications, mainly in non-veterinary journals, have argued for the need for appropriate validation of the reference genes used to report relative expression levels of specific gene products with accuracy. It is abundantly clear that calculations of gene expression levels relative to a single, unvalidated, reference gene, without any stability expression evaluation, can be highly misleading (Dheda et al., 2005). A quick survey of papers published in The Veterinary Journal in 2009 and 2010 revealed only two papers in which several reference genes were used to calculate relative gene expression. Veronica Spalenza and colleagues at the University of Turin evaluated several reference genes in bovine peripheral lymphocytes (Spalenza et al., 2011). In a paper by Eric Zini and colleagues at the Vetsuisse Faculty, University of Zurich, three reference genes were used for relative gene expression calculations (Zini et al., 2010). Very few veterinary publications provide information on RNA quality and integrity and there is little information on the reverse transcription step or on the efficiency of the subsequent PCR. This makes it difficult to evaluate the relevance of any data reported in these journals or to attempt to reproduce the data in a different laboratory. Standardisation of reporting procedures and, indeed, reporting of a minimum amount of relevant technical information of molecular strategies is of paramount importance if gene expression studies are to be more reliable. The so-called ‘Minimum Information for publication of Quantitative real-time PCR Experiments’ (MIQE) guidelines aim to improve the standard of publications utilising qPCR by providing a checklist of the critical information required to enhance readers’ appreciation of the data, facilitate the repeatability of these experiments and enhance the comparison between different studies (Bustin et al., 2009). These guidelines are gradually being put into practice, with more than 300 citations appearing in the peer-reviewed literature in the last year; their general adoption by veterinary researchers would serve to strengthen the research field of veterinary medicine. A recently published modified standardisation approach, MIQEprecis (Bustin et al., 2010) offers simplified guidelines and a Microsoft Excel-based checklist aimed at improving transparency and 1090-0233/$ see front matter ! 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.06.044 ⇑ Corresponding author. Tel.: +31 3