Abstract 3491: Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR.

AbstractPotential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

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

Food Control ◽

10.1016/j.foodcont.2014.06.018 ◽

2014 ◽

Vol 46 ◽

pp. 470-474 ◽

Cited By ~ 21

Author(s):

Tigst Demeke ◽

Tom Gräfenhan ◽

Michelle Holigroski ◽

Ursla Fernando ◽

Janice Bamforth ◽

...

Keyword(s):

Digital Pcr ◽

Absolute Quantification ◽

Genetically Engineered ◽

Droplet Digital Pcr

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Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽

10.1161/hyp.68.suppl_1.p613 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Ryousuke Satou ◽

Akemi Katsurada ◽

Kayoko Miyata ◽

Andrei Derbenev ◽

Andrea Zsombok

Keyword(s):

Copy Number ◽

System Analysis ◽

Renin Angiotensin System ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Angiotensin System ◽

Renin Angiotensin ◽

Copy Numbers ◽

Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

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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 ◽

10.3390/w12123507 ◽

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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Droplet Digital PCR for Absolute Quantification of Extracellular MicroRNAs in Plasma and Serum: Quantification of the Cancer Biomarker hsa-miR-141

Methods in Molecular Biology - Digital PCR ◽

10.1007/978-1-4939-7778-9_26 ◽

2018 ◽

pp. 459-474 ◽

Cited By ~ 5

Author(s):

Maria D. Giraldez ◽

John R. Chevillet ◽

Muneesh Tewari

Keyword(s):

Digital Pcr ◽

Absolute Quantification ◽

Cancer Biomarker ◽

Droplet Digital Pcr

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Abstract 4956: Droplet digital PCR in validating copy number variations in hereditary prostate cancer families from Louisiana

10.1158/1538-7445.am2016-4956 ◽

2016 ◽

Author(s):

Kirsten Wood ◽

Ratish Gambhira ◽

Elisa Ledet ◽

Oliver Sartor ◽

Diptasri Mandal

Keyword(s):

Prostate Cancer ◽

Copy Number ◽

Digital Pcr ◽

Copy Number Variations ◽

Droplet Digital Pcr ◽

Hereditary Prostate Cancer

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Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control

Genes ◽

10.3390/genes10030243 ◽

2019 ◽

Vol 10 (3) ◽

pp. 243 ◽

Cited By ~ 10

Author(s):

Teruaki Tozaki ◽

Aoi Ohnuma ◽

Masaki Takasu ◽

Mio Kikuchi ◽

Hironaga Kakoi ◽

...

Keyword(s):

Magnetic Beads ◽

Genetic Manipulation ◽

Digital Pcr ◽

Absolute Quantification ◽

Droplet Digital Pcr ◽

Taqman Probe ◽

Gene Doping ◽

Athletic Ability ◽

Horse Plasma ◽

Primer Sets

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.

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