Tania Nolan

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

BACKGROUND Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a readers ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

Quantification of mRNA using real-time RT-PCR

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a “gold” standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer.

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene ‘hyperplastic discs’ and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT–PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Minimum information necessary for quantitative real-time PCR experiments.

The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information inadequate for the purpose of assessing the validity of published qPCR data. This had, and continues to have serious implications for basic research, reducing the potential for translating findings into valuable applications and potentially devastating consequences for clinical practice. Today, the rationale underlying the MIQE guidelines has become widely accepted, with more than 2,200 citations by March 2014 and editorials in Nature and related publications acknowledging the enormity of the problem. However, the problem we now face is rather serious: thousands of publications that report suspect data are populating and corrupting the peer-reviewed scientific literature. It will be some time before the many contradictions apparent in every area of the life sciences are corrected.

Variability of the Reverse Transcription Step: Practical Implications

BACKGROUND The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability. METHODS We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay. RESULTS RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed. CONCLUSIONS Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

Improving the reliability of peer-reviewed publications: We are all in it together

The current, and welcome, focus on standardization of techniques and transparency of reporting in the biomedical, peer-reviewed literature is commendable. However, that focus has been intermittent as well as lacklustre and so failed to tackle the alarming lack of reliability and reproducibly of biomedical research. Authors have access to numerous recommendations, ranging from simple standards dealing with technical issues to those regulating clinical trials, suggesting that improved reporting guidelines are not the solution. The elemental solution is for editors to require meticulous implementation of their journals’ instructions for authors and reviewers and stipulate that no paper is published without a transparent, complete and accurate materials and methods section.

Chapter 9:Procedures for Quality Control of RNA Samples for Use in Quantitative Reverse Transcription PCR

The quality of any scientific data is directly proportional to that of the original starting samples, or simply ‘garbage in, garbage out’. In most circumstances it is logical to work with the highest quality material possible. However, for some experiments the highest quality possible is still a ser...