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Digital PCR

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https://www.readbyqxmd.com/read/28213308/simultaneous-estimation-of-detection-sensitivity-and-absolute-copy-number-from-digital-pcr-serial-dilution
#1
Xutao Deng, Brian S Custer, Michael P Busch, Sonia Bakkour, Tzong-Hae Lee
Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets...
February 1, 2017: Computational Biology and Chemistry
https://www.readbyqxmd.com/read/28212714/droplet-based-digital-pcr-application-in-cancer-research
#2
G Perkins, H Lu, F Garlan, V Taly
The efficient characterization of genetic and epigenetic alterations in oncology, virology, or prenatal diagnostics requires highly sensitive and specific high-throughput approaches. Nevertheless, with the use of conventional methods, sensitivity and specificity were largely limited. By partitioning individual target molecules within distinct compartments, digital PCR (dPCR) could overcome these limitations and detect very rare sequences with unprecedented precision and sensitivity. In dPCR, the sample is diluted such that each individual partition will contain no more than one target sequence...
2017: Advances in Clinical Chemistry
https://www.readbyqxmd.com/read/28209368/correlation-of-micro-vessel-density-and-c-myc-expression-in-breast-tumor-of-mice-following-mesenchymal-stem-cell-therapy
#3
Maryam Adelipour, Fatemeh Babaei, Mohammadreza Mirzababaei, Abdolamir Allameh
Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I...
January 30, 2017: Tissue & Cell
https://www.readbyqxmd.com/read/28205231/analysis-of-ctdna-to-predict-prognosis-and-monitor-treatment-responses-in-metastatic-pancreatic-cancer-patients
#4
He Cheng, Chen Liu, Jiahao Jiang, Guopei Luo, Yu Lu, Kaizhou Jin, Meng Guo, Zhenzhen Zhang, Jin Xu, Liang Liu, Quanxing Ni, Xianjun Yu
Cell-free circulating tumor DNA (ctDNA) in plasma has been used as a potential noninvasive biomarker for various tumors. The current study was performed to evaluate the clinical implications of ctDNA detection in patients with metastatic pancreatic cancer. Firstly, we attempted to prospectively screen a panel of 60 genes in cell-free DNA (cfDNA) from ten metastatic pancreatic cancer patients via exome sequencing. Secondly, droplet digital PCR (ddPCR) was used to identify potential mutations in a cohort of 188 patients with metastatic pancreatic cancer...
February 16, 2017: International Journal of Cancer. Journal International du Cancer
https://www.readbyqxmd.com/read/28197972/prevalence-and-molecular-characterization-of-the-hepatitis-e-virus-in-retail-pork-products-marketed-in-canada
#5
Oksana Mykytczuk, Jennifer Harlow, Sabah Bidawid, Nathalie Corneau, Neda Nasheri
Infection with the hepatitis E virus (HEV) is very common worldwide. HEV causes acute viral hepatitis with approximately 20 million cases per year. While HEV genotypes 1 and 2 cause large waterborne and foodborne outbreaks with a significant mortality in developing countries, genotypes 3 and 4 are more prevalent in developed countries with transmission being mostly zoonotic. In North America and Europe, HEV has been increasingly detected in swine, and exposure to pigs and pork products is considered to be the primary source of infection...
February 14, 2017: Food and Environmental Virology
https://www.readbyqxmd.com/read/28196522/xpo1-in-b-cell-hematological-malignancies-from-recurrent-somatic-mutations-to-targeted-therapy
#6
REVIEW
Vincent Camus, Hadjer Miloudi, Antoine Taly, Brigitte Sola, Fabrice Jardin
Many recent publications highlight the large role of the pivotal eukaryotic nuclear export protein exportin-1 (XPO1) in the oncogenesis of several malignancies, and there is emerging evidence that XPO1 inhibition is a key target against cancer. The clinical validation of the pharmacological inhibition of XPO1 was recently achieved with the development of the selective inhibitor of nuclear export compounds, displaying an interesting anti-tumor activity in patients with massive pre-treated hematological malignancies...
February 14, 2017: Journal of Hematology & Oncology
https://www.readbyqxmd.com/read/28193205/comparison-of-immunohistochemistry-with-pcr-for-assessment-of-er-pr-and-ki-67-and-prediction-of-pathological-complete-response-in-breast-cancer
#7
Hans-Peter Sinn, Andreas Schneeweiss, Marius Keller, Kornelia Schlombs, Mark Laible, Julia Seitz, Sotirios Lakis, Elke Veltrup, Peter Altevogt, Sebastian Eidt, Ralph M Wirtz, Frederik Marmé
BACKGROUND: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. METHODS: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial...
February 13, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28193149/taqman-probes-as-blocking-agents-for-enriched-pcr-amplification-and-dna-melting-analysis-of-mutant-genes
#8
Irina V Botezatu, Irina O Panchuk, Anna M Stroganova, Anastasia I Senderovich, Valentina N Kondratova, Valery P Shelepov, Anatoly V Lichtenstein
Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28191690/mutational-profiling-of-acral-melanomas-in-korean-populations
#9
Joon Ho Shim, Hyun-Tae Shin, Jiho Park, Ji-Hye Park, Jong-Hee Lee, Jun-Mo Yang, Duk-Hwan Kim, Kee-Taek Jang, Dong-Youn Lee
The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasians populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in the Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profilinge of AM and matched normal tissues of in patients. Fifty-one formalin-fixed paraffin-embedded AM samples and 32 matched pairs of from patients' saliva DNA were analyzed by next-generation sequencing...
February 13, 2017: Experimental Dermatology
https://www.readbyqxmd.com/read/28191267/single-cell-digital-polymerase-chain-reaction-on-self-priming-compartmentalization-chip
#10
Qiangyuan Zhu, Lin Qiu, Yanan Xu, Guang Li, Ying Mu
Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell...
January 2017: Biomicrofluidics
https://www.readbyqxmd.com/read/28190454/somatic-map2k1-mutations-are-associated-with-extracranial-arteriovenous-malformation
#11
Javier A Couto, August Y Huang, Dennis J Konczyk, Jeremy A Goss, Steven J Fishman, John B Mulliken, Matthew L Warman, Arin K Greene
Arteriovenous malformation (AVM) is a fast-flow, congenital vascular anomaly that may arise anywhere in the body. AVMs typically progress, causing destruction of surrounding tissue and, sometimes, cardiac overload. AVMs are difficult to control; they often re-expand after embolization or resection, and pharmacologic therapy is unavailable. We studied extracranial AVMs in order to identify their biological basis. We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) on AVM tissue from affected individuals...
January 30, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28188954/comparative-glandular-trichome-transcriptome-based-gene-characterization-reveals-reasons-for-differential-menthol-biosynthesis-in-mentha-species
#12
Md Qussen Akhtar, Nida Qamar, Pallavi Yadav, Pallavi Kulkarni, Ajay Kumar, Ajit Kumar Shasany
The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome was carried out. In addition to the MVA and MEP pathway genes, about 210 and 196 different terpene synthases (TPS) transcripts were identified from annotation in M...
February 11, 2017: Physiologia Plantarum
https://www.readbyqxmd.com/read/28183140/correlation-between-pik3ca-mutations-in-cell-free-dna-and-everolimus-efficacy-in-hr-her2-advanced-breast-cancer-results-from-bolero-2
#13
Mary Ellen Moynahan, David Chen, Wei He, Patricia Sung, Aliaksandra Samoila, Daoqi You, Trusha Bhatt, Parul Patel, Francois Ringeisen, Gabriel N Hortobagyi, Jose Baselga, Sarat Chandarlapaty
BACKGROUND: The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation. METHODS: PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm. RESULTS: Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0...
February 9, 2017: British Journal of Cancer
https://www.readbyqxmd.com/read/28177428/monitoring-of-somatic-mutations-in-circulating-cell-free-dna-by-digital-pcr-and-next-generation-sequencing-during-afatinib-treatment-in-patients-with-lung-adenocarcinoma-positive-for-egfr-activating-mutations
#14
E Iwama, K Sakai, K Azuma, T Harada, D Harada, K Nosaki, K Hotta, F Ohyanagi, T Kurata, T Fukuhara, H Akamatsu, K Goto, T Shimose, J Kishimoto, Y Nakanishi, K Nishio, I Okamoto
No abstract text is available yet for this article.
January 1, 2017: Annals of Oncology: Official Journal of the European Society for Medical Oncology
https://www.readbyqxmd.com/read/28176860/massively-parallel-digital-high-resolution-melt-for-rapid-and-absolutely-quantitative-sequence-profiling
#15
Daniel Ortiz Velez, Hannah Mack, Julietta Jupe, Sinead Hawker, Ninad Kulkarni, Behnam Hedayatnia, Yang Zhang, Shelley Lawrence, Stephanie I Fraley
In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously...
February 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28174565/evaluation-of-molecular-methods-for-the-detection-and-quantification-of-pathogen-derived-nucleic-acids-in-sediment
#16
Kata Farkas, Francis Hassard, James E McDonald, Shelagh K Malham, Davey L Jones
The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution-concentration) methods were evaluated...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28173668/-epidermal-growth-factor-receptor-egfr-mutation-status-before-and-after-acquired-resistance-to-egfr-tyrosine-kinase-inhibitors-in-patients-with-lung-adenocarcinoma
#17
S B Ye, R Li, S S Shi
Objective: To investigate epidermal growth factor receptor (EGFR) mutation status in lung adenocarcinomas before and after acquiring resistance to EGFR tyrosine kinase inhibitors (TKIs) using ARMS method followed by further verification using droplet digital PCR technique. Methods: Twenty qualified patients were included, among them 13 were male and 7 were female patients. Before EGFR-TKIs treatment, 5 patients were EGFR wild-type by ARMS, and the other 15 patients had L858R or 19-del point mutations. The time to progression varied from 4 to 18 months...
February 8, 2017: Zhonghua Bing Li Xue za Zhi Chinese Journal of Pathology
https://www.readbyqxmd.com/read/28164617/evaluation-of-labtype%C3%A2-sso-hla-typing-using-the-luminex-platform-cord-blood-registry-typing-for-the-korean-population
#18
Eun-Youn Roh, Eun-Young Song, Jee-Young Chang, Jong-Hyun Yoon, Sue Shin
BACKGROUND: The performance of a new intermediate-resolution method using a PCR-Luminex platform and LABType® SSO A, B DRB1 kits as an HLA typing method for the cord blood (CB) registry of the Korean population was investigated. METHODS: A total of 1,413 cord blood units (CBUs) were enrolled - 1,382 from Koreans and 31 from non-Koreans or mixed-ancestry individuals. HLA-A, -B, and -DRB1 typing was performed using the LABType® SSO typing kits. HLA typing with the DNA method and 2-digit results are mandatory for the public CB bank in Korea according to the "CB Act...
August 1, 2016: Clinical Laboratory
https://www.readbyqxmd.com/read/28164515/performance-of-lifecodes-hla-a-b-and-drb1-typing-kits-using-luminex-platform-in-koreans
#19
Eun Youn Roh, Ji Won In, Sue Shin, Kyoung Un Park, Eun Young Song
BACKGROUND: Recently, HLA typing using PCR sequence-specific oligonucleotide (SSO) and the Luminex platform has been introduced and widely used in clinical laboratories. METHODS: We evaluated the performance of LIFECODES HLA-A, -B, and -DRB1 typing kits (Immucor, USA) on 108 samples that had been tested with WAKFlow kits (Wakunaga, Japan) and 54 samples (18 for each A, B, and DRB1 kits) including rare alleles in Koreans (gene frequency < 1.0%) that had been assigned by sequence-based typing (SBT)...
October 1, 2016: Clinical Laboratory
https://www.readbyqxmd.com/read/28164427/application-of-circulating-tumor-dna-in-prospective-clinical-oncology-trials-standardization-of-pre-analytical-conditions
#20
Lisanne F van Dessel, Nick Beije, Jean C A Helmijr, Silvia R Vitale, Jaco Kraan, Maxime P Look, Ronald de Wit, Stefan Sleijfer, Maurice P H M Jansen, John W M Martens, Martijn P J K Lolkema
Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various pre-analytical conditions on the quality of cell-free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant...
February 6, 2017: Molecular Oncology
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