Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

2017 ◽  
Vol 96 ◽  
pp. 339-344 ◽  
Author(s):  
Zhenhua Wu ◽  
Yanan Bai ◽  
Zule Cheng ◽  
Fangming Liu ◽  
Ping Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Microfluidic Chip ◽  
Digital Pcr ◽  
Absolute Quantification

scholarly journals A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR

Micromachines ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1025
Author(s):  
Gangwei Xu ◽  
Huaqing Si ◽  
Fengxiang Jing ◽  
Peng Sun ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Mutual Interference ◽  
Fluorescence Signal ◽  
Pdms Layer ◽  
Glass Substrates ◽  
Planar Area ◽  
Dna Template

In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets. The double-deck designs can double the number of chambers and reagent capacity without changing the planar area of the chip. In addition, carbon black was mixed into the pure PDMS gel to obstruct the passage of fluorescence from the positive chambers between the two layers of the chip. Thus, the fluorescence signal of micro-chambers in different layers of the chip after PCR can be collected without mutual interference. The quantitative capability of the proposed chip was evaluated by measuring a 10-fold serial dilution of the DNA template. A high accuracy of the absolute quantification for nucleic acid with a dynamic range of 105 was demonstrated by this chip in this work. Owing to its characteristics of small planar area, large capacity, and sensitivity, the double-deck microfluidic chip is expected to further promote the extensive applications of digital PCR.

scholarly journals A Self-Priming Microfluidic Chip with Cushion Chambers for Easy Digital PCR

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 158
Author(s):  
Gangwei Xu ◽  
Huaqing Si ◽  
Fengxiang Jing ◽  
Peng Sun ◽  
Dongping Wu
Keyword(s):  
Microfluidic Chip ◽  
Low Cost ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Pdms Layer ◽  
Resource Limited ◽  
Dna Template ◽  
Digital Polymerase Chain Reaction ◽  
Easy Operation

A polydimethylsiloxane (PDMS)-based self-priming microfluidic chip with cushion chambers is presented in this study for robust and easy-operation digital polymerase chain reaction (dPCR). The chip has only one inlet and can partition samples autonomously through negative pressure, provided by a de-gassed PDMS layer with a multi-level vertical branching microchannel design. Meanwhile, cushion chambers make the chip capable of very robust use for sample partitioning. Finally, the proposed microfluidic chip showed excellent performance in the absolute quantification of a target gene by performing quantitative detection of a 10-fold serial dilution DNA template. Owing to its characteristics of easy operation, low cost, and high robustness, the proposed dPCR chip is expected to further promote the extensive application of digital PCR, especially in resource-limited settings.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

scholarly journals Sepsis Diagnostics: Intensive Care Scoring Systems Superior to MicroRNA Biomarker Testing

Diagnostics ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 701
Author(s):  
Fabian Link ◽  
Knut Krohn ◽  
Anna-Maria Burgdorff ◽  
Annett Christel ◽  
Julia Schumann
Keyword(s):  
Intensive Care ◽  
Scoring Systems ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Medical Problem ◽  
Control Group ◽  
Multiple Parameters ◽  
Sepsis Diagnosis ◽  
Intensive Care Patients ◽  
Healthy Control

Sepsis represents a serious medical problem accounting for numerous deaths of critically ill patients in intensive care units (ICUs). An early, sensitive, and specific diagnosis is considered a key element for improving the outcome of sepsis patients. In addition to classical laboratory markers, ICU scoring systems and serum miRNAs are discussed as potential sepsis biomarkers. In the present prospective observational study, the suitability of miRNAs in sepsis diagnosis was tested based on proper validated and normalized data (i.e., absolute quantification by means of Droplet Digital PCR (ddPCR)) in direct comparison to classical sepsis markers and ICU scores within the same patient cohort. Therefore, blood samples of septic intensive care patients (n = 12) taken at day of admission at ICU were compared to non-septic intensive care patients (n = 12) and a healthy control group (n = 12). Our analysis indicates that all tested biomarkers have only a moderate informative power and do not allow an unequivocal differentiation between septic and non-septic ICU patients. In conclusion, there is no standalone laboratory parameter that enables a reliable diagnosis of sepsis. miRNAs are not superior to classical parameters in this respect. It seems recommendable to measure multiple parameters and scores and to interpret them with regard to the clinical presentation.

Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples

Food Control ◽  
2014 ◽  
Vol 46 ◽  
pp. 470-474 ◽  
Author(s):  
Tigst Demeke ◽  
Tom Gräfenhan ◽  
Michelle Holigroski ◽  
Ursla Fernando ◽  
Janice Bamforth ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Droplet Digital Pcr

scholarly journals Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Water ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3507
Author(s):  
Mark A. Ibekwe ◽  
Shelton E. Murinda ◽  
Stanley Park ◽  
Amarachukwu Obayiuwana ◽  
Marcia A. Murry ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Point Of Care ◽  
Foodborne Pathogen ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
High Rate ◽  
E Coli

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

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