Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment.

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling.

Meta-Analysis of qPCR for Bovine Respiratory Disease Based on MIQE Guidelines.

Qualitative and quantitative PCR-based tests are widely used in both diagnostics and research to assess the prevalence of disease-causing pathogens in veterinary medicine. The efficacy of these tests, usually measured in terms of sensitivity and specificity, is critical in confirming or excluding a clinical diagnosis. We undertook a meta-analysis to assess the inherent value of published PCR diagnostic approaches used to confirm and quantify bacteria and viruses associated with bovine respiratory disease (BRD) in cattle. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A thorough search of nine electronic databases (Web of Science, EBSCOhost, Cambridge journals online, ProQuest, PubMed, Sage journals online, ScienceDirect, Wiley online library and MEDLINE) was undertaken to find studies that had reported on the use of PCR and/or qPCR for the detection and/or quantification of BRD associated organisms. All studies meeting the inclusion criteria for reporting quantitative PCR for identification of BRD associated microorganisms were included in the analysis. Studies were then assessed on the applications of the Minimum Information for Publication of Quantitative Real-Time PCR Experiment (MIQE) and PCR primer/probe sequences were extracted and tested for in silico specificity using a high level of stringency. Fourteen full-text articles were included in this study. Of these, 79% of the analysed articles did not report the application of the MIQE guidelines in their study. High stringency in silico testing of 144 previously published PCR primer/probe sequences found many to have questionable specificity. This review identified a high occurrence of primer/probe sequences with a variable in silico specificity such that this may have implications for the accuracy of reporting. Although this analysis was only applied to one specific disease state, identification of animals suspected to be suffering from bovine respiratory disease, there appears to be more broadly a need for veterinary diagnostic studies to adopt international best practice for reporting of quantitative PCR diagnostic data to be both accurate and comparable between studies and methodologies.

Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) for Gene Expression Analyses.

The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is considered to be the gold standard for gene expression research. However, for this claim to be valid, RT-qPCR studies must test and optimize the quality of its RNA templates and assays. This chapter describes the experimental procedures required to generate reliable and reproducible gene expression results using RT-qPCR.

Assessment of human nuclear and mitochondrial DNA qPCR assays for quantification accuracy utilizing NIST SRM 2372a.

In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male1 donor, Component B) a single female1 donor, and Component C) a 1:3 male2:female2 donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted.

Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics-MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery.

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of "key steps" in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

Standardization of a high-performance RT-qPCR for viral load absolute quantification of influenza A.

Influenza is an acute viral infectious respiratory disease worldwide, presenting in different clinical forms, from influenza-like illness (ILI) to severe acute respiratory infection (SARI). Although real-time quantitative polymerase chain reaction (qPCR) is already an important tool for both diagnosis and treatment monitoring of several viral infections, the correlation between the clinical aspects and the viral load of influenza is still unclear. This lack of clarity is primarily due to the low accuracy and reproducibility of the methodologies developed to quantify the influenza virus. Thus, this study aimed to develop and standardize a universal absolute quantification for influenza A by reverse transcription-quantitative PCR (RT-qPCR), using a plasmid DNA. The assay showed efficiency (Eff%) 98.6, determination coefficient (R2) 0.998, linear range 10^1 to 10^10, limit of detection (LOD) 6.77, limit of quantification (LOQ) 20.52 copies/reaction. No inter and intra assay variability was shown, and neither was the matrix effect observed. Serial measurements of clinical samples collected at a 72h interval showed no change in viral load. By contrast, immunocompetent patients have a significantly lower viral load than immunosuppressed ones. Absolute quantification in clinical samples showed some predictors associated with increased viral load: (H1N1)pdm09 (0.045); women (p = 0.049) and asthmatics (p = 0.035). The high efficiency, precision, and previous performance in clinical samples suggest the assay can be used as an accurate universal viral load quantification of influenza A. Its applicability in predicting severity and response to antivirals needs to be evaluated.

Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction.

Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.

RT-qPCR for Detection and Expression Monitoring of Core Genes Involved in the RNA Interference Pathways of Western Corn Rootworm (Diabrotica virgifera virgifera).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays are a highly accurate and precise method for measuring transcript expression levels. A major drawback of RT-qPCR is the extensive optimization and validation necessary to produce high-quality assays, as described in the guidelines "Minimum Information for Publication of Quantitative Real-Time PCR Experiments." This chapter describes use of designed and optimized RT-qPCR assays that accurately detect expression of eight genes predicted to be centrally involved in the RNA interference (RNAi) pathways of western corn rootworm (WCR), and appropriate accompanying parameters. Assay gene targets include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2, and detection has been validated at nine different points in the WCR life cycle. These assays can be used with this procedure to assess expression of any one of these core RNAi pathway genes in up to 96 samples per 384-well qPCR plate.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults.

BACKGROUND: Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. METHODS: A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. RESULTS: The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. CONCLUSIONS: The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

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.

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020.

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

Parameters for Successful PCR Primer Design.

Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous guidelines, the actual design of many published assays is often unsound: primers lack the claimed specificity, they may have to compete with secondary structures at their binding sites, primer dimer formation may affect the assay's sensitivity or they may bind only within a narrow temperature range. This chapter provides simple guidance to avoid these most common issues.

MIQE-Compliant Validation of MicroRNA Biomarker Signatures Established by Small RNA Sequencing.

MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression at the post-transcriptional level, are attractive targets in many academic and diagnostic applications. Among them, assessing miRNA biomarkers in minimally invasive liquid biopsies was shown to be a promising tool for managing diseases, particularly cancer. The initial screening of disease-relevant transcripts is often performed by high-throughput next-generation sequencing (NGS), in here RNA sequencing (RNA-Seq). After complex processing of small RNA-Seq data, differential gene expression analysis is performed to evaluate miRNA biomarker signatures. To ensure experimental validity, biomarker candidates are commonly validated by an orthogonal technology such as reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). This chapter outlines in detail the material and methods one can apply to reproducibly identify miRNA biomarker signatures from blood total RNA. After screening miRNA profiles by small RNA-Seq, resulting data is validated in compliance with the "Minimum Information for Publication of Quantitative Real-Time PCR Experiments" (MIQE) guidelines.

ELIMU-MDx: a web-based, open-source platform for storage, management and analysis of diagnostic qPCR data.

The Electronic Laboratory Information and Management Utensil for Molecular Diagnostics (ELIMU-MDx) is a user-friendly platform designed and built to accelerate the turnaround time of diagnostic qPCR assays. ELIMU-MDx is compliant with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and has extensive data-import capabilities for all major qPCR instruments by using the RDML data standard. This platform was designed as an open-source software tool and can be accessed through the web browser on all major operating systems.

SATQPCR: Website for statistical analysis of real-time quantitative PCR data.

SATQPCR is a web tool providing statistical analysis of real-time quantitative PCR data including all MIQE rules (gene efficiency, selection of reference genes and normalization with them). Our application is a quick tool that provides to the biologist, graphs as well as statistical tables summarizing their results with the chosen methods (t-test or ANOVA with Tukey test). The application is available at http://satqpcr.sophia.inra.fr with a demo dataset. Source code can be found at https://framagit.org/. SUPPLEMENTARY INFORMATION: Tutorials at http://satqpcr.sophia.inra.fr/cgi/help.cgi.

siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR.

The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20-25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.

The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time.

Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR.

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested.