Category Ranking
98%
Total Visits
921
Avg Visit Duration
2 minutes
Citations
20
Article Abstract
Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both with suitable calibration curves and efficiencies. We demonstrated thereby the benefits of PCR-Stop analysis revealing quantitative and qualitative resolution of both assays, the limits of one of those assays and thus avoiding misinterpretations in qPCR analysis. Furthermore, data displayed that a well performing assay starts indeed with its average efficiency.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974342 | PMC |
| http://dx.doi.org/10.1038/s41598-018-26116-x | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Attenuation of c-Myc expression in breast cancer by hesperidin-mediated stabilization of its promoter proximal G quadruplex region.
Int J Biol Macromol
May 2025
Department of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology, Patiala, Punjab 147004, India; Centre for Excellence in Emerging Materials, Thapar Institute of Engineering and Technology, Patiala, Punjab 147004, India. Electronic address:
Hesperidin, a citrus flavanone, demonstrates significant potential as an anticancer agent by targeting the c-Myc G-quadruplex (G4) silencer element (Pu-27), a key epigenetic regulator of c-Myc expression. Molecular docking analysis revealed a strong interaction with Pu-27 (binding energy: -48.344 kcal/mol), forming hydrogen bonds across five critical regions.
View Article and Find Full Text PDFA Novel G-Quadruplex Structure within Apolipoprotein E Promoter: A New Promising Target in Cancer and Dementia Fight?
ACS Omega
November 2024
Istituto di Genetica Molecolare "Luigi Luca Cavalli-Sforza", via Abbiategrasso 207, I-27100 Pavia, Italy.
Human apolipoprotein E (APOE) is a crucial lipid transport glycoprotein involved in various biological processes, including lipid metabolism, immune response, and neurodegeneration. Elevated APOE levels are linked to poor prognosis in several cancers and increased risk of Alzheimer's disease (AD). Therefore, modulating APOE expression presents a promising therapeutic strategy for both cancer and AD.
View Article and Find Full Text PDFRatio of the Primers Used in Polymerase Chain Reaction-Stop Analysis Impacts the Resultant Banding Pattern.
ACS Omega
October 2023
Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
G-quadruplex (G4), as a dynamic nucleic acid secondary structure, widely exists in organism genomes and plays regulatory roles in a variety of cellular functions. Polymerase chain reaction stop assay (PCR-Stop) is a simple, quick, and low-cost widely used method for detection of the binding between G4 and its binding compounds. Different from the common PCR approach, no double-stranded DNA template is needed in the PCR-Stop assay, in which the forward and reverse primers extend against each other in the presence of DNA polymerase to produce a single DNA product.
View Article and Find Full Text PDFqPCR Validation on the Basis of the Listeria monocytogenes prfA Assay.
Methods Mol Biol
April 2021
Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria.
Quantitative real-time polymerase chain reaction (qPCR) is one of the most used molecular methods. There are numerous qPCR assays on the market, some of them for pathogen detection, and the development of new assays still continues. However, what methods are suitable for assay performance validation and which information do they provide? For conclusions based on qPCR data, it is essential to know which capacities and limitations an assay has.
View Article and Find Full Text PDFEssential role of polymerases for assay performance - Impact of polymerase replacement in a well-established assay.
Biomol Detect Quantif
December 2018
Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinaerplatz 1, 1210, Vienna, Austria.
The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations.
View Article and Find Full Text PDF