Inhibition of phosphodiesterases 1 and 4 prevents myofibroblast transformation in Peyronie's disease.

Objectives: To investigate which phosphodiesterase (PDE) isoforms are expressed in fibroblasts isolated from the tunica albuginea (TA) of patients with Peyronie's disease (PD), and to measure the potency of PDE inhibitors in preventing transformation of these fibroblasts to profibrotic myofibroblasts.

Materials And Methods: Fibroblasts isolated from the TA of men undergoing surgery for correction of PD curvature were transformed to myofibroblasts using transforming growth factor beta-1. The expression of 21 PDE isoforms was investigated using quantitative reverse transcriptase-polymerase chain reaction and protein analysis, as were the effects of various PDE inhibitors on prevention of myofibroblast transformation.

RT-qPCR Testing and Performance Metrics in the COVID-19 Era.

The COVID-19 pandemic highlighted the crucial role of diagnostic testing in managing infectious diseases, particularly through the use of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests. RT-qPCR has been pivotal in detecting and quantifying viral RNA, enabling the identification and management of SARS-CoV-2 infections. However, despite its widespread use, there remains a notable gap in understanding fundamental diagnostic metrics such as sensitivity and specificity among many scientists and healthcare practitioners.

TGF-β1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation.

Extracellular vesicles have emerged as important mediators of cell-to-cell communication in the pathophysiology of fibrotic diseases. One such disease is Peyronie's disease (PD), a fibrotic disorder of the penis caused by uncontrolled transformation of resident fibroblasts to alpha-smooth muscle actin positive myofibroblasts. These cells produce large amounts of extracellular matrix, leading to formation of a plaque in the penile tunica albuginea (TA), causing pain, penile curvature, and erectile dysfunction.

FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols.

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices.

Temporal gene signature of myofibroblast transformation in Peyronie's disease: first insights into the molecular mechanisms of irreversibility.

Background: Transformation of resident fibroblasts to profibrotic myofibroblasts in the tunica albuginea is a critical step in the pathophysiology of Peyronie's disease (PD). We have previously shown that myofibroblasts do not revert to the fibroblast phenotype and we suggested that there is a point of no return at 36 hours after induction of the transformation. However, the molecular mechanisms that drive this proposed irreversibility are not known.

Improving the quality of quantitative polymerase chain reaction experiments: 15 years of MIQE.

The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics.

Advances in Molecular Medicine: Unravelling Disease Complexity and Pioneering Precision Healthcare.

The escalating impacts of the climate crisis, zoonotic spill-over, and antibiotic resistance have positioned molecular medicine at the forefront of pioneering translational research [...

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.

Molecular Pathology, Diagnostics and Therapeutics: A Story of Success in 2022.

Molecular pathology, diagnostics and therapeutics are three closely related topics of critical importance in medical research and clinical practice [...

Maximising the Use of Scarce qPCR Master Mixes.

The COVID-19 pandemic resulted in a universal, immediate, and vast demand for comprehensive molecular diagnostic testing, especially real-time quantitative (qPCR)-based methods. This rapidly triggered a global shortage of testing capacity, equipment, and reagents. Even today, supply times for chemicals from date of order to delivery are often much longer than pre-pandemic.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay.

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.

RT-qPCR Diagnostics: The "Drosten" SARS-CoV-2 Assay Paradigm.

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence.

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance.

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols.

COVID-19 and Diagnostic Testing for SARS-CoV-2 by RT-qPCR-Facts and Fallacies.

Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.

CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19.

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity.

Cautionary Note on Contamination of Reagents Used for Molecular Detection of SARS-CoV-2.

Reverse transcription (RT)-PCR, the principal diagnostic method applied in the world-wide struggle against COVID-19, is capable of detecting a single molecule of a viral genome. Correctly designed and practiced RT-PCR assays for SARS-CoV-2 should not cross react with similar but distinct viral pathogens, such as the coronaviruses associated with the common cold, and should perform with very high analytical sensitivity. This analytical performance is predicated on the ability of the method to detect the presence of the selected nucleic acid target, without detection of a false positive signal.

Phosphodiesterase Type 5 Inhibitors and Selective Estrogen Receptor Modulators Can Prevent But Not Reverse Myofibroblast Transformation in Peyronie's Disease.

Background: Myofibroblast transformation is a key step in the pathogenesis of Peyronie's disease (PD). Phosphodiesterase type 5 inhibitors (PDE5is) and selective estrogen receptor modulators (SERMs) can prevent the formation of fibrosis in in vitro and in vivo models of PD. However, it is unknown whether these drugs can also reverse established fibrosis.

RT-qPCR Testing of SARS-CoV-2: A Primer.

Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability.

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.

Improving the standardization of mRNA measurement by RT-qPCR.

Human health and safety depend on reliable measurements in medical diagnosis and on tests that support the selection and evaluation of therapeutic intervention and newly discovered molecular biomarkers must pass a rigorous evaluation process if they are to be of benefit to patients. Measurement standardization helps to maximize data quality and confidence and ultimately improves the reproducibility of published research. Failure to consider how a given experiment may be standardized can be costly, both financially as well as in time and failure to perform and report pre-clinical research in an appropriately rigorous manner will hinder the development of diagnostic methods.

Evaluation of the new AspID polymerase chain reaction assay for detection of Aspergillus species: A pilot study.

The newly developed AspID PCR assay for detection of Aspergillus spp. was evaluated with an interlaboratory quality control programme panel and human bronchoalveolar lavage fluid (BALF) samples. With the quality control programme, 8 out of 9 panel members were correctly identified.

qPCR primer design revisited.

Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range.

Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research.

Poorly executed and inadequately reported molecular measurement methods are amongst the causes underlying the lack of reproducibility of much biomedical research. Although several high impact factor journals have acknowledged their past failure to scrutinise adequately the technical soundness of manuscripts, there is a perplexing reluctance to implement basic corrective measures. The reverse transcription real-time quantitative PCR (RT-qPCR) is probably the most straightforward measurement technique available for RNA quantification and is widely used in research, diagnostic, forensic and biotechnology applications.

How to speed up the polymerase chain reaction.

Reducing the time taken to run qPCR assays on today's qPCR cyclers is rather straightforward and requires no specialised reagents or instruments. As the first article in a new series of short technical reports, I demonstrate that it is possible to reduce significantly both denaturation temperatures and cycling times, whilst retaining sensitivity and specificity of the original qPCR conditions.