Five years MIQE guidelines: The case of the Arabian countries

The quantitative real time polymerase chain reaction (qPCR) has become a key molecular enabling technology with an immense range of research, clinical, forensic as well as diagnostic applications. Its relatively moderate instrumentation and reagent requirements have led to its adoption by numerous laboratories, including those located in the Arabian world, where qPCR, which targets DNA, and reverse transcription qPCR (RT-qPCR), which targets RNA, are widely used for region-specific biotechnology, agricultural and human genetic studies. However, it has become increasingly apparent that there are significant problems with both the quality of qPCR-based data as well as the transparency of reporting. This realisation led to the publication of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines in 2009 and their more widespread adoption in the last couple of years. An analysis of the performance of biomedical research in the Arabian world between 2001-2005 suggests that the Arabian world is producing fewer biomedical publications of lower quality than other Middle Eastern countries. Hence we have analysed specifically the quality of RT-qPCR-based peer-reviewed papers published since 2009 from Arabian researchers using a bespoke iOS/Android app developed by one of the authors. Our results show that compliance with 15 essential MIQE criteria was low (median of 40%, range 0-93%) and few details on RNA quality controls (22% compliance), assays design (12%), RT strategies (32%), amplification efficiencies (30%) and the normalisation process (3%). These data indicate that one of the reasons for the poor performance of Arabian world biomedical research may be the low standard of any supporting qPCR experiments and identify which aspects of qPCR experiments require significant improvements

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been us Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and N Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Assessment of myocardial injuries in ischemic and non-ischemic cardiomyopathies using magnetic resonance T1-rho mapping.

To identify clinical correlates of myocardial T1ρ and to examine how myocardial T1ρ values change under various clinical scenarios. A total of 66 patients (26% female, median age 57 years [Q1-Q3, 44-65 years]) with known structural heart disease and 44 controls (50% female, median age 47 years [28-57 years]) underwent cardiac magnetic resonance imaging at 1.5-T, including T1ρ mapping, T2 mapping, native T1 mapping, late gadolinium enhancement, and ECV imaging.In controls, T1ρ positively related with T2 (P=0.038) and increased from basal to apical levels (P<0.001). As compared to controls and remote myocardium, T1ρ significantly increased in all patients' sub-groups and all types of myocardial injuries: acute and chronic injuries, focal and diffuse tissue abnormalities, as well as ischemic and non-ischemic aetiologies (P<0.05). T1ρ was independently associated with T2 in patients with acute injuries (P=0.004) and with native T1 and ECV in patients with chronic injuries (P<0.05). Myocardial T1ρ mapping demonstrated good intra- and interobserver reproducibility (ICC=0.86 and 0.83, respectively). Myocardial T1ρ mapping appears to be reproducible and equally sensitive to acute and chronic myocardial injuries, whether of ischemic or non-ischemic origins. It may thus be a contrast-agent-free biomarker for gaining new and quantitative insight into myocardial structural disorders. These findings highlight the need for further studies through prospective and randomized trials

A versatile panel of reference gene assays for the measurement of chicken mRNA by quantitative PCR

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology

Theoretical analysis of the role of chromatin interactions in long-range action of enhancers and insulators

Long-distance regulatory interactions between enhancers and their target genes are commonplace in higher eukaryotes. Interposed boundaries or insulators are able to block these long distance regulatory interactions. The mechanistic basis for insulator activity and how it relates to enhancer action-at-a-distance remains unclear. Here we explore the idea that topological loops could simultaneously account for regulatory interactions of distal enhancers and the insulating activity of boundary elements. We show that while loop formation is not in itself sufficient to explain action at a distance, incorporating transient non-specific and moderate attractive interactions between the chromatin fibers strongly enhances long-distance regulatory interactions and is sufficient to generate a euchromatin-like state. Under these same conditions, the subdivision of the loop into two topologically independent loops by insulators inhibits inter-domain interactions. The underlying cause of this effect is a suppression of crossings in the contact map at intermediate distances. Thus our model simultaneously accounts for regulatory interactions at a distance and the insulator activity of boundary elements. This unified model of the regulatory roles of chromatin loops makes several testable predictions that could be confronted with \emph{in vitro} experiments, as well as genomic chromatin conformation capture and fluorescent microscopic approaches.Comment: 10 pages, originally submitted to an (undisclosed) journal in May 201

Magnetic Resonance Fingerprinting using Recurrent Neural Networks

Magnetic Resonance Fingerprinting (MRF) is a new approach to quantitative magnetic resonance imaging that allows simultaneous measurement of multiple tissue properties in a single, time-efficient acquisition. Standard MRF reconstructs parametric maps using dictionary matching and lacks scalability due to computational inefficiency. We propose to perform MRF map reconstruction using a recurrent neural network, which exploits the time-dependent information of the MRF signal evolution. We evaluate our method on multiparametric synthetic signals and compare it to existing MRF map reconstruction approaches, including those based on neural networks. Our method achieves state-of-the-art estimates of T1 and T2 values. In addition, the reconstruction time is significantly reduced compared to dictionary-matching based approaches.Comment: Accepted for ISBI 201

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study

BACKGROUND: Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. RESULTS: The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. CONCLUSIONS: Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens

Impact of Reference Gene Selection for Target Gene Normalization on Experimental Outcome Using Real-Time qRT-PCR in Adipocytes

Background: With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., b-actin, GAPDH, a-tubulin) are differentially regulated across multiple tissue types and experimental conditions. Methodology/Principal Findings: Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. Conclusion/Significance: Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significan