Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Web-based LinRegPCR: application for the visualization and analysis of (RT)-qPCR amplification and melting data.

BACKGROUND: The analyses of amplification and melting curves have been shown to provide valuable information on the quality of the individual reactions in quantitative PCR (qPCR) experiments and to result in more reliable and reproducible quantitative results. IMPLEMENTATION: The main steps in the amplification curve analysis are (1) a unique baseline subtraction, not using the ground phase cycles, (2) PCR efficiency determination from the exponential phase of the individual reactions, (3) setting a common quantification threshold and (4) calculation of the efficiency-corrected target quantity with the common threshold, efficiency per assay and Cq per reaction. The melting curve analysis encompasses smoothing of the observed fluorescence data, normalization to remove product-independent fluorescence loss, peak calling and assessment of the correct peak by comparing its melting temperature with the known melting temperature of the intended amplification product. RESULTS: The LinRegPCR web application provides visualization and analysis of a single qPCR run. The user interface displays the analysis results on the amplification curve analysis and melting curve analysis in tables and graphs in which deviant reactions are highlighted. The annotated results in the tables can be exported for calculation of gene-expression ratios, fold-change between experimental conditions and further statistical analysis. Web-based LinRegPCR addresses two types of users, wet-lab scientists analyzing the amplification and melting curves of their own qPCR experiments and bioinformaticians creating pipelines for analysis of series of qPCR experiments by splitting its functionality into a stand-alone back-end RDML (Real-time PCR Data Markup Language) Python library and several companion applications for data visualization, analysis and interactive access. The use of the RDML data standard enables machine independent storage and exchange of qPCR data and the RDML-Tools assist with the import of qPCR data from the files exported by the qPCR instrument. CONCLUSIONS: The combined implementation of these analyses in the newly developed web-based LinRegPCR ( https://www.gear-genomics.com/rdml-tools/ ) is platform independent and much faster than the original Windows-based versions of the LinRegPCR program. Moreover, web-based LinRegPCR includes a novel statistical outlier detection and the combination of amplification and melting curve analyses allows direct validation of the amplification product and reporting of reactions that amplify artefacts.

Use and Misuse of Cq in qPCR Data Analysis and Reporting.

In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.

Efficiency Correction Is Required for Accurate Quantitative PCR Analysis and Reporting.

BACKGROUND: Quantitative PCR (qPCR) aims to measure the DNA or RNA concentration in diagnostic and biological samples based on the quantification cycle (Cq) value observed in the amplification curves. Results of qPCR experiments are regularly calculated as if all assays are 100% efficient or reported as just Cq, ΔCq, or ΔΔCq values. CONTENTS: When the reaction shows specific amplification, it should be deemed to be positive, regardless of the observed Cq. Because the Cq is highly dependent on amplification efficiency that can vary among targets and samples, accurate calculation of the target quantity and relative gene expression requires that the actual amplification efficiency be taken into account in the analysis and reports. PCR efficiency is frequently derived from standard curves, but this approach is affected by dilution errors and hampered by properties of the standard and the diluent. These factors affect accurate quantification of clinical and biological samples used in diagnostic applications and collected in challenging conditions. PCR efficiencies determined from individual amplification curves avoid these confounders. To obtain unbiased efficiency-corrected results, we recommend absolute quantification with a single undiluted calibrator with a known target concentration and efficiency values derived from the amplification curves of the calibrator and the unknown samples. SUMMARY: For meaningful diagnostics or biological interpretation, the reported results of qPCR experiments should be efficiency corrected. To avoid ambiguity, the Minimal Information for Publications on Quantitative Real-Time PCR Experiments (MIQE) guidelines checklist should be extended to require the methods that were used (1) to determine the PCR efficiency and (2) to calculate the reported target quantity and relative gene expression value.

Removal of artifact bias from qPCR results using DNA melting curve analysis.

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter, J. M., Ruiz-Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis.

Colourimetric analysis of thermally altered human bone samples.

At this moment, no method is available to objectively estimate the temperature to which skeletal remains have been exposed during a fire. Estimating this temperature can provide crucial information in a legal investigation. Exposure of bone to heat results in observable and measurable changes, including a change in colour. To determine the exposure temperature of experimental bone samples, heat related changes in colour were systemically studied by means of image analysis. In total 1138 samples of fresh human long bone diaphysis and epiphysis, varying in size, were subjected to heat ranging from room temperature to 900 °C for various durations and in different media. The samples were scanned with a calibrated flatbed scanner and photographed with a Digital Single Lens Reflex camera. Red, Green, Blue values and Lightness, A-, and B-coordinates were collected for statistical analysis. Cluster analysis showed that discriminating thresholds for Lightness and B-coordinate could be defined and used to construct a model of decision rules. This model enables the user to differentiate between seven different temperature clusters with relatively high precision and accuracy. The proposed decision model provides an objective, robust and non-destructive method for estimating the exposure temperature of heated bone samples.

Considerations for Measurement of Embryonic Organ Growth.

Organogenesis is a complex coordinated process of cell proliferation, growth, migration, and apoptosis. Differential growth rates, particularly during cardiogenesis, play a role in establishing morphology. Studies using stereological and cell sorting methods derive averages of morphogenetic parameters for an organ. To understand tissue composition and differential growth, the researcher must determine a number of morphogenetic parameters in the developing organ. Such measurements require sectioning to enable identification of organ borders, tissue components and cell types, three-dimensional (3D)-reconstruction of sections to visualize morphology and a 3D-measurement scheme to build local morphogenetic information. Although thick the section confocal microscopy partially solves these issues, information loss at the section surface hampers the reconstruction of 3D morphology. Episcopic imaging provides the correct morphology but lacks histological procedures to identify multiple cell types. The 3D-measurement scheme is based on systematic sampling, with overlapping sample volumes, of the entire organ in the aligned image stack. For each sample volume, morphogenetic variables are calculated and results projected back to the cube (boxel) at the sample volume center. Boxel size determines spatial resolution of the final quantitative 3D-reconstruction whereas size of the sample volume determines the precision of the morphogenetic information. The methods described here can be used to measure tissue volume, proliferation and cell size, to determine contribution and distribution of cell types in a tissue and to display this information in a quantitative 3D-reconstruction. Anat Rec, 302:49-57, 2019. © 2018 Wiley Periodicals, Inc.

Interfering With DNA Decondensation as a Strategy Against Mycobacteria.

Tuberculosis is once again a major global threat, leading to more than 1 million deaths each year. Treatment options for tuberculosis patients are limited, expensive and characterized by severe side effects, especially in the case of multidrug-resistant forms. Uncovering novel vulnerabilities of the pathogen is crucial to generate new therapeutic strategies. Using high resolution microscopy techniques, we discovered one such vulnerability of Mycobacterium tuberculosis. We demonstrate that the DNA of M. tuberculosis can condense under stressful conditions such as starvation and antibiotic treatment. The DNA condensation is reversible and specific for viable bacteria. Based on these observations, we hypothesized that blocking the recovery from the condensed state could weaken the bacteria. We showed that after inducing DNA condensation, and subsequent blocking of acetylation of DNA binding proteins, the DNA localization in the bacteria is altered. Importantly under these conditions, Mycobacterium smegmatis did not replicate and its survival was significantly reduced. Our work demonstrates that agents that block recovery from the condensed state of the nucleoid can be exploited as antibiotic. The combination of fusidic acid and inhibition of acetylation of DNA binding proteins, via the Eis enzyme, potentiate the efficacy of fusidic acid by 10 and the Eis inhibitor to 1,000-fold. Hence, we propose that successive treatment with antibiotics and drugs interfering with recovery from DNA condensation constitutes a novel approach for treatment of tuberculosis and related bacterial infections.

Paradoxical effects of mutant ubiquitin on Aß plaque formation in an Alzheimer mouse model.

Amyloid-ß (Aß) plaques are a prominent pathological hallmark of Alzheimer's disease (AD). They consist of aggregated Aß peptides, which are generated through sequential proteolytic processing of the transmembrane protein amyloid precursor protein (APP) and several Aß-associated factors. Efficient clearance of Aß from the brain is thought to be important to prevent the development and progression of AD. The ubiquitin-proteasome system (UPS) is one of the major pathways for protein breakdown in cells and it has been suggested that impaired UPS-mediated removal of protein aggregates could play an important role in the pathogenesis of AD. To study the effects of an impaired UPS on Aß pathology in vivo, transgenic APPSwe/PS1ΔE9 mice (APPPS1) were crossed with transgenic mice expressing mutant ubiquitin (UBB+1), a protein-based inhibitor of the UPS. Surprisingly, the APPPS1/UBB+1 crossbreed showed a remarkable decrease in Aß plaque load during aging. Further analysis showed that UBB+1 expression transiently restored PS1-NTF expression and γ-secretase activity in APPPS1 mice. Concurrently, UBB+1 decreased levels of ß-APP-CTF, which is a γ-secretase substrate. Although UBB+1 reduced Aß pathology in APPPS1 mice, it did not improve the behavioral deficits in these animals.

Study Design and qPCR Data Analysis Guidelines for Reliable Circulating miRNA Biomarker Experiments: A Review.

BACKGROUND: In the past decade, the search for circulating microRNA (miRNA) biomarkers has yielded numerous associations between miRNAs and different types of disease. However, many of these relations could not be replicated in subsequent studies under similar experimental conditions. Although this lack of replicability may be explained by the variation in experimental design and analysis methods, guidelines on the most appropriate design and analysis methods to study circulating miRNAs are scarce. CONTENT: miRNA biomarker experiments generally consist of a discovery phase and a validation phase. In the discovery phase, typically hundreds of miRNAs are measured in parallel to identify candidate biomarkers. Because of the costs of such high-throughput experiments, the number of individuals included in those studies is often too small, which can easily lead to false positives and false negatives. In the validation phase, a small number of identified biomarker candidates are measured in a large cohort of cases and controls, generally by quantitative PCR (qPCR). Although qPCR is a sensitive method to measure miRNAs in the circulation, experimental design and qPCR data analysis remain challenging. Omitting some crucial steps in the design and analysis of the qPCR experiment or performing them incorrectly can cause serious biases, ultimately leading to false conclusions. SUMMARY: In this review, we aim to expose and discuss the most common sources of interstudy variation in miRNA research from a methodological point of view and to provide guidelines on how to perform these steps correctly to increase replicability of studies on circulating miRNAs.

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR).

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template -plasmid DNA- and non-template -mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.

Maternal diabetes causes developmental delay and death in early-somite mouse embryos.

Maternal diabetes causes congenital malformations and delays embryonic growth in the offspring. We investigated effects of maternal diabetes on mouse embryos during gastrulation and early organogenesis (ED7.5-11.5). Female mice were made diabetic with streptozotocin, treated with controlled-release insulin implants, and mated. Maternal blood glucose concentrations increased up to embryonic day (ED) 8.5. Maternal hyperglycemia induced severe growth retardation (approx.1 day) in 53% of the embryos on ED8.5, death in most of these embryos on ED9.5, and the termination of pregnancy on ED10.5 in litters with >20% dead embryos. Due to this selection, developmental delays and reduction in litter size were no longer observed thereafter in diabetic pregnancies. Male and female embryos were equally sensitive. High-throughput mRNA sequencing and pathway analysis of differentially expressed genes showed that retarded embryos failed to mount the adaptive suppression of gene expression that characterized non-retarded embryos (cell proliferation, cytoskeletal remodeling, oxidative phosphorylation). We conclude that failure of perigastrulation embryos of diabetic mothers to grow and survive is associated with their failure to shut down pathways that are strongly down-regulated in otherwise similar non-retarded embryos. Embryos that survive the early and generalized adverse effect of maternal diabetes, therefore, appear the subset in which malformations become manifest.

Deletion of Fstl1 (Follistatin-Like 1) From the Endocardial/Endothelial Lineage Causes Mitral Valve Disease.

OBJECTIVE: Fstl1 (Follistatin-like 1) is a secreted protein that is expressed in the atrioventricular valves throughout embryonic development, postnatal maturation, and adulthood. In this study, we investigated the loss of Fstl1 in the endocardium/endothelium and their derived cells. APPROACH AND RESULTS: We conditionally ablated Fstl1 from the endocardial lineage using a transgenic Tie2-Cre mouse model. These mice showed a sustained Bmp and Tgfß signaling after birth. This resulted in ongoing proliferation and endocardial-to-mesenchymal transition and ultimately in deformed nonfunctional mitral valves and a hypertrophic dilated heart. Echocardiographic and electrocardiographic analyses revealed that loss of Fstl1 leads to mitral regurgitation and left ventricular diastolic dysfunction. Cardiac function gradually deteriorated resulting in heart failure with preserved ejection fraction and death of the mice between 2 and 4 weeks after birth. CONCLUSIONS: We report on a mouse model in which deletion of Fstl1 from the endocardial/endothelial lineage results in deformed mitral valves, which cause regurgitation, heart failure, and early cardiac death. The findings provide a potential molecular target for the clinical research into myxomatous mitral valve disease.

The effect of repeated freeze-thaw cycles on human muscle tissue visualized by postmortem computed tomography (PMCT).

The aim of this study was to determine whether effects of repetitive freeze-thaw cycles, with various thawing temperatures, on human muscle tissue can be quantified using postmortem computed tomography (PMCT) technology. An additional objective was to determine the preferred thawing temperature for muscle tissue in this study. Human cadaver upper extremities were divided into two different thawing temperature groups and underwent a series of four freeze-thaw cycles in total. Axial CT scans were performed after each cycle. CT attenuation (in Hounsfield units, HU) was measured in four muscles of the upper extremities. HU values changed significantly with the introduction of each subsequent freeze-thaw cycle. Moreover, the changes in HU values were different for each thawing group. There was a significant increase of HU values in both groups between t0 and t1 . Unfrozen tissue showed large variation of HU values in all samples. It was possible to distinguish between samples thawed at different thawing temperatures based on their respective HU values. It is advisable to keep the number of freeze-thaw cycles to just one, if the human cadaveric tissue is to be used for educational purposes. The preferred thawing temperature in this study is 2°C. Clin. Anat. 30:799-804, 2017. © 2017Wiley Periodicals, Inc.

Reference genes for gene expression studies in the mouse heart.

To be accurate, quantitative Polymerase Chain Reaction (qPCR) studies require a set of stable reference genes for normalization. This is especially critical in cardiac research because of the diversity of the clinical and experimental conditions in the field. We analyzed the stability of previously described as potential reference genes in different subsets of cardiac tissues, each representing a different field in cardiac research. The qPCR dataset was based on 119 different tissue samples derived from cardiac development to pathology in mouse adult hearts. These samples were grouped into 47 tissue types. The stability of 9 candidate genes was analyzed in each of 12 experimental conditions comprising different groupings of these tissue types. Expression stability was determined with the geNorm module of qbase+. This analysis showed that different sets of two or three reference genes are required for analysis of qPCR data in different experimental conditions in murine cardiac research.

The geometrical precision of virtual bone models derived from clinical computed tomography data for forensic anthropology.

Almost all European countries lack contemporary skeletal collections for the development and validation of forensic anthropological methods. Furthermore, legal, ethical and practical considerations hinder the development of skeletal collections. A virtual skeletal database derived from clinical computed tomography (CT) scans provides a potential solution. However, clinical CT scans are typically generated with varying settings. This study investigates the effects of image segmentation and varying imaging conditions on the precision of virtual modelled pelves. An adult human cadaver was scanned using varying imaging conditions, such as scanner type and standard patient scanning protocol, slice thickness and exposure level. The pelvis was segmented from the various CT images resulting in virtually modelled pelves. The precision of the virtual modelling was determined per polygon mesh point. The fraction of mesh points resulting in point-to-point distance variations of 2 mm or less (95% confidence interval (CI)) was reported. Colour mapping was used to visualise modelling variability. At almost all (>97%) locations across the pelvis, the point-to-point distance variation is less than 2 mm (CI = 95%). In >91% of the locations, the point-to-point distance variation was less than 1 mm (CI = 95%). This indicates that the geometric variability of the virtual pelvis as a result of segmentation and imaging conditions rarely exceeds the generally accepted linear error of 2 mm. Colour mapping shows that areas with large variability are predominantly joint surfaces. Therefore, results indicate that segmented bone elements from patient-derived CT scans are a sufficiently precise source for creating a virtual skeletal database.

Practical data handling pipeline improves performance of qPCR-based circulating miRNA measurements.

Since numerous miRNAs have been shown to be present in circulation, these so-called circulating miRNAs have emerged as potential biomarkers for disease. However, results of qPCR studies on circulating miRNA biomarkers vary greatly and many experiments cannot be reproduced. Missing data in qPCR experiments often occur due to off-target amplification, nonanalyzable qPCR curves and discordance between replicates. The low concentration of most miRNAs leads to most, but not all missing data. Therefore, failure to distinguish between missing data due to a low concentration and missing data due to randomly occurring technical errors partly explains the variation within and between otherwise similar studies. Based on qPCR kinetics, an analysis pipeline was developed to distinguish missing data due to technical errors from missing data due to a low concentration of the miRNA-equivalent cDNA in the PCR reaction. Furthermore, this pipeline incorporates a method to statistically decide whether concentrations from replicates are sufficiently concordant, which improves stability of results and avoids unnecessary data loss. By going through the pipeline's steps, the result of each measurement is categorized as "valid, invalid, or undetectable." Together with a set of imputation rules, the pipeline leads to more robust and reproducible data as was confirmed experimentally. Using two validation approaches, in two cohorts totaling 2214 heart failure patients, we showed that this pipeline increases both the accuracy and precision of qPCR measurements. In conclusion, this statistical data handling pipeline improves the performance of qPCR studies on low-expressed targets such as circulating miRNAs.

Luminescence of thermally altered human skeletal remains.

Literature on luminescent properties of thermally altered human remains is scarce and contradictory. Therefore, the luminescence of heated bone was systemically reinvestigated. A heating experiment was conducted on fresh human bone, in two different media, and cremated human remains were recovered from a modern crematory. Luminescence was excited with light sources within the range of 350 to 560 nm. The excitation light was filtered out by using different long pass filters, and the luminescence was analysed by means of a scoring method. The results show that temperature, duration and surrounding medium determine the observed emission intensity and bandwidth. It is concluded that the luminescent characteristic of bone can be useful for identifying thermally altered human remains in a difficult context as well as yield information on the perimortem and postmortem events.

Pivotal role of glutamine synthetase in ammonia detoxification.

Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that ∼35% of this ammonia is detoxified by hepatic GS and ∼35% by urea-cycle enzymes, while ∼30% is not cleared by the liver, independent of portal ammonia concentrations ≤2 mmol/L. Using both genetic (GS-knockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is ∼160 µmol/hour in control mice. Using stable isotopes, we show that disposal of glutamine-bound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from ∼7% in methionine sulfoximine-treated mice to ∼500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. CONCLUSIONS: Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (Hepatology 2017;65:281-293).