Keeping qRT-PCR rigorous and biologically relevant

Autor: James C. Register, John Bennett, David Hondred
Rok vydání: 2014
Předmět:
Zdroj: Plant cell reports. 34(1)
ISSN: 1432-203X
Popis: Because of the importance of differential gene expression in the growth, development, and reproduction of organisms, assays of mRNA transcript levels are widely employed in biological research. The most frequently used assay combines reverse transcription (RT) with quantitative polymerase chain reaction (qPCR) and is known as qRT-PCR (or RT-qPCR). In practice, RT generates a single-stranded cDNA copy of each molecule in an mRNA population, after which a qPCR thermocycler is used in conjunction with gene-specific oligonucleotide primers to amplify selected target cDNAs (Bustin et al. 2009). Fluorescent probes allow progress to be monitored through three phases: an initial phase during which the increasing signal cannot be distinguished from background noise, a 2nd phase of observable exponential signal increase, and a third and final phase of slowing signal increase as reagents and other factors become rate limiting. Quantification of each target mRNA requires the determination of the number of cycles (known as Ct, Cq, Cp, or TOP) required to reach a pre-set fluorescence threshold during the second phase. When a transcript is twice as abundant in one sample as it is in another, the Ct will occur about one cycle earlier for the more abundant transcript. The varying transcript levels of target genes must then be normalized against the stable transcript levels of one or more reference genes present in the same mRNA population. Algorithms are available to validate the stability of these reference genes. With use of appropriate controls, standardization, and normalization, measurements of Ct can lead to reliable estimates of the relative amount of a target transcript in the input mRNA population (Bustin et al. 2009). Despite its complexity, qRT-PCR is rapid and cost effective, and, if sufficient care is taken with the design and execution of experiments, it is reliable and sensitive. There are, however, two trends of concern in the publication of qRT-PCR experiments. The first is that many papers do not contain enough information to judge whether the data are reliable. Bustin et al. (2009) addressed this question by defining ‘‘minimum information for publication of quantitative real-time PCR experiments’’ (MIQE). The second trend of concern is the increasing number of manuscripts that claim to have identified reference genes without comparing them to target genes in a well-defined and relevant biological context. We agree with Bustin et al. (2010), who considered it pointless and misleading to publish such ‘reference gene papers’ as stand-alone reports. In response to these trends, we propose that publishable qRT-PCR manuscripts should satisfy two criteria. First, the design, execution, and reporting of qRT-PCR experiments should address the items listed in the MIQE guidelines. Second, the selection and validation of reference genes should be reported in relation to target gene behavior in a well-defined biological context. Here we discuss the background to these criteria and their implementation relative to plant biology studies. Communicated by Neal Stewart.
Databáze: OpenAIRE
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