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https://www.readbyqxmd.com/read/29021970/quantification-of-mitochondrial-dna-copy-number-in-suspected-cancer-patients-by-a-well-optimized-ddpcr-method
#1
Ashfaque A Memon, Bengt Zöller, Anna Hedelius, Xiao Wang, Emelie Stenman, Jan Sundquist, Kristina Sundquist
Changes in mitochondrial DNA (mtDNA) content is a useful clinical biomarker for various diseases, however results are controversial as several analytical factors can affect measurement of mtDNA. MtDNA is often quantified by taking ratio between a target mitochondrial gene and a reference nuclear gene (mtDNA/nDNA) using quantitative real time PCR often on two separate experiments. It measures relative levels by using external calibrator which may not be comparable across laboratories. We have developed and optimized a droplet digital PCR (ddPCR) based method for quantification of absolute copy number of both mtDNA and nDNA gene in whole blood...
September 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/29019928/the-abundance-of-toxic-genotypes-is-a-key-contributor-to-anatoxin-variability-in-phormidium-dominated-benthic-mats
#2
Susanna A Wood, Jonathan Puddick
The prevalence of benthic proliferations of the anatoxin-producing cyanobacterium Phormidium are increasing in cobble-bed rivers worldwide. Studies to date have shown high spatial and temporal variability in anatoxin concentrations among mats. In this study we determined anatoxin quotas (toxins per cell) in field samples and compared these results to the conventionally-used concentrations (assessed per dry weight of mat). Three mats were selected at sites in two rivers and were sampled every 2-3 h for 24-26 h...
October 11, 2017: Marine Drugs
https://www.readbyqxmd.com/read/29018576/key-differences-between-13-kras-mutation-detection-technologies-and-their-relevance-for-clinical-practice
#3
James L Sherwood, Helen Brown, Alessandro Rettino, Amelie Schreieck, Graeme Clark, Bart Claes, Bhuwnesh Agrawal, Ria Chaston, Benjamin S G Kong, Paul Choppa, Anders O H Nygren, Ina L Deras, Alexander Kohlmann
INTRODUCTION: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner. METHODS: Five distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays...
2017: ESMO Open
https://www.readbyqxmd.com/read/28986787/copy-number-variation-analysis-by-droplet-digital-pcr
#4
Suvi K Härmälä, Robert Butcher, Chrissy H Roberts
The health impact of many copy number variants in our genome remains still largely to be discovered. Detecting and genotyping this often complex variation presents a technical challenge. Here we describe a 96-well format droplet digital PCR (ddPCR) protocol for genotyping a common copy variant in the human haptoglobin gene. ddPCR allows for high-throughput and accurate quantitation of gene copy numbers.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28966190/12-year-use-of-a-digital-reference-library-vitalbook-at-a-u-s-dental-school-students-and-alumni-perceptions
#5
Andrew I Spielman, Elizabeth Maas, Elise S Eisenberg
Digital textbooks are being used to reduce production and storage costs of printed copies, enhance usage, and include search capabilities, but the use of digital texts is not universally accepted. In 2001, the New York University College of Dentistry introduced a digital reference library, the VitalBook. Beginning in 2005, the college annually surveyed senior students and, from 2012, also surveyed alumni on their opinions and extent of use of the VitalBook. The aim of this study was to evaluate 12 years of students' perspectives and three years of alumni perspectives on the value of the VitalBook to their dental educational experience...
October 2017: Journal of Dental Education
https://www.readbyqxmd.com/read/28957377/androgen-receptor-expression-on-circulating-tumor-cells-in-metastatic-breast-cancer
#6
Takeo Fujii, James M Reuben, Lei Huo, Jose Rodrigo Espinosa Fernandez, Yun Gong, Rachel Krupa, Mahipal V Suraneni, Ryon P Graf, Jerry Lee, Stephanie Greene, Angel Rodriguez, Lyndsey Dugan, Jessica Louw, Bora Lim, Carlos H Barcenas, Angela N Marx, Debu Tripathy, Yipeng Wang, Mark Landers, Ryan Dittamore, Naoto T Ueno
PURPOSE: Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer...
2017: PloS One
https://www.readbyqxmd.com/read/28956373/-progress-in-digital-pcr-technology-and-application
#7
Jiaqi Lin, Guocheng Su, Wenjin Su, Changyi Zhou
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food...
February 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28951374/-rapid-detection-of-alpha-globin-gene-%C3%AE-%C3%AE-%C3%AE-anti-3-7-triplets-with-droplet-digital-pcr
#8
Xiao-Qian Gong, Xue-Huang Yang, Lin-Li Qiao, Ya-Jun Cheng, Wan-Jun Zhou
OBJECTIVE: To establish a rapid method for detection of alpha-globin gene ααα(anti-3.7) based on droplet digital PCR (ddPCR) technique. METHODS: The differential sequence between the X1 and Y1 box of α1 gene was selected as the amplicon of the target gene with β-actin as the reference gene. The specific primers and TaqMan probes were designed, and then a quantitative method for detecting the copy number was established based on ddPCR technique. The sensitivity and accuracy of the method were evaluated by detecting 28 samples of known genotypes and 60 clinical samples...
September 20, 2017: Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University
https://www.readbyqxmd.com/read/28928368/detection-of-clinically-relevant-copy-number-alterations-in-oral-cancer-progression-using-multiplexed-droplet-digital-pcr
#9
Curtis B Hughesman, X J David Lu, Kelly Y P Liu, Yuqi Zhu, Rebecca M Towle, Charles Haynes, Catherine F Poh
Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays...
September 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28923315/development-of-a-digital-droplet-pcr-assay-to-measure-hbv-dna-in-patients-receiving-long-term-tdf-treatment
#10
Yang Liu, Andrea L Cathcart, William E Delaney, Kathryn M Kitrinos
The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (<58 copies/mL) or target detected (TD) (between 58 and 169 copies/mL). Here we have developed a more sensitive digital droplet PCR (ddPCR) assay to evaluate the impact of long-term tenofovir disoproxil fumarate (TDF) treatment in patients that did or did not achieve HBsAg seroconversion. A ddPCR assay was developed to detect HBV DNA to 8 copies/mL...
September 18, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28921124/droplet-volume-variability-as-a-critical-factor-for-accuracy-of-absolute-quantification-using-droplet-digital-pcr
#11
Alexandra Bogožalec Košir, Carla Divieto, Jernej Pavšič, Stefano Pavarelli, David Dobnik, Tanja Dreo, Roberto Bellotti, Maria Paola Sassi, Jana Žel
Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies...
September 18, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28915237/dna-replication-stress-restricts-ribosomal-dna-copy-number
#12
Devika Salim, William D Bradford, Amy Freeland, Gillian Cady, Jianmin Wang, Steven C Pruitt, Jennifer L Gerton
Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes...
September 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28910375/application-of-droplet-digital-pcr-for-quantitative-detection-of-spiroplasma-citri-in-comparison-with-real-time-pcr
#13
Yogita Maheshwari, Vijayanandraj Selvaraj, Subhas Hajeri, Raymond Yokomi
Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S...
2017: PloS One
https://www.readbyqxmd.com/read/28902411/functional-changes-in-the-neural-retina-occur-in-the-absence-of-mitochondrial-dysfunction-in-a-rodent-model-of-diabetic-retinopathy
#14
Dustin R Masser, Laura Otalora, Nicholas W Clark, Michael T Kinter, Michael H Elliott, Willard M Freeman
Diabetic retinopathy is a neurovascular diabetes complication resulting in vision loss. A wealth of literature reports retinal molecular changes indicative of neural deficits, inflammation, and vascular leakage with chronic diabetes, but the mechanistic causes of disease initiation and progression are unknown. Microvascular mitochondrial DNA (mtDNA) damage leading to mitochondrial dysfunction has been proposed to drive vascular dysfunction in retinopathy. However, growing evidence suggests that neural retina dysfunction precedes and may cause vascular damage...
September 13, 2017: Journal of Neurochemistry
https://www.readbyqxmd.com/read/28893733/topological-analysis-reveals-a-pd-l1-associated-microenvironmental-niche-for-reed-sternberg-cells-in-hodgkin-lymphoma
#15
Christopher D Carey, Daniel Gusenleitner, Mikel Lipschitz, Margaretha G M Roemer, Edward C Stack, Evisa Gjini, Xihao Hu, Robert Redd, Gordon J Freeman, Donna Neuberg, F Stephen Hodi, Xiaole Shirley Liu, Margaret A Shipp, Scott J Rodig
Signaling between programmed cell death protein 1 (PD-1) and the programmed cell death -1 ligands (PD-1 ligands, PD-L1, PD-L2) is essential for malignant Hodgkin Reed-Sternberg (HRS) cells to evade anti-tumor immunity in classical Hodgkin lymphoma (cHL). Copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) contribute to robust PD-L1 and PD-L2 expression by HRS cells. PD-L1 is also expressed by non-malignant tumor-associated macrophages (TAMs) but the relationships between PD-L1+ HRS cells, PD-L1+ TAMs, and PD-1+ T-cells remain undefined...
September 11, 2017: Blood
https://www.readbyqxmd.com/read/28846100/composition-and-dosage-of-a-multipartite-enhancer-cluster-control-developmental-expression-of-ihh-indian-hedgehog
#16
Anja J Will, Giulia Cova, Marco Osterwalder, Wing-Lee Chan, Lars Wittler, Norbert Brieske, Verena Heinrich, Jean-Pierre de Villartay, Martin Vingron, Eva Klopocki, Axel Visel, Darío G Lupiáñez, Stefan Mundlos
Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips...
October 2017: Nature Genetics
https://www.readbyqxmd.com/read/28841507/copy-move-forgery-detection-through-stationary-wavelets-and-local-binary-pattern-variance-for-forensic-analysis-in-digital-images
#17
Toqeer Mahmood, Aun Irtaza, Zahid Mehmood, Muhammad Tariq Mahmood
The most common image tampering often for malicious purposes is to copy a region of the same image and paste to hide some other region. As both regions usually have same texture properties, therefore, this artifact is invisible for the viewers, and credibility of the image becomes questionable in proof centered applications. Hence, means are required to validate the integrity of the image and identify the tampered regions. Therefore, this study presents an efficient way of copy-move forgery detection (CMFD) through local binary pattern variance (LBPV) over the low approximation components of the stationary wavelets...
October 2017: Forensic Science International
https://www.readbyqxmd.com/read/28827685/absolute-measurement-of-gene-transcripts-with-selfie-digital-pcr
#18
Petar Podlesniy, Ramon Trullas
Absolute measurement of the number of RNA transcripts per gene is necessary to compare gene transcription among different tissues or experimental conditions and to assess transcription of genes that have a variable copy number per cell such as mitochondrial DNA. Here, we present a method called Selfie-digital PCR that measures the absolute amount of an RNA transcript produced by its own coding DNA at a particular moment. Overcoming the limitations of previous approaches, Selfie-digital PCR allows for the quantification of nuclear and mitochondrial gene transcription in a strand-specific manner that is comparable among tissues and cell types that differ in gene copy number or metabolic state...
August 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28826609/a-droplet-digital-pcr-method-for-severe-combined-immunodeficiency-newborn-screening
#19
Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S Abraham, Devin Oglesbee
Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch...
September 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28823057/fusobacterium-nucleatum-as-a-prognostic-marker-of-colorectal-cancer-in-a-japanese-population
#20
Yuko Yamaoka, Yutaka Suehiro, Shinichi Hashimoto, Tomomi Hoshida, Michiyo Fujimoto, Michiya Watanabe, Daiki Imanaga, Kouhei Sakai, Toshihiko Matsumoto, Mitsuaki Nishioka, Taro Takami, Nobuaki Suzuki, Shoichi Hazama, Hiroaki Nagano, Isao Sakaida, Takahiro Yamasaki
BACKGROUND: Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer. METHODS: We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues...
August 19, 2017: Journal of Gastroenterology
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