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https://www.readbyqxmd.com/read/28231382/accurate-measurement-of-transgene-copy-number-in-crop-plants-using-droplet-digital-pcr
#1
Ray Collier, Kasturi Dasgupta, Yan-Ping Xing, Bryan Tarape Hernandez, Min Shao, Dominica Rohozinski, Emma Kovak, Jeanie Lin, Maria Luiza P de Oliveira, Ed Stover, Kent F McCue, Frank G Harmon, Ann Blechl, James G Thomson, Roger Thilmony
Genetic transformation is a powerful means for the improvement of crop plants, but requires labor and resource intensive methods. An efficient method for identifying single copy transgene insertion events from a population of independent transgenic lines is desirable. Currently transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes...
February 23, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28228143/relationship-between-salivary-pancreatic-amylase-and-body-mass-index-a-systems-biology-approach
#2
Amélie Bonnefond, Loïc Yengo, Aurélie Dechaume, Mickaël Canouil, Maxime Castelain, Estelle Roger, Frédéric Allegaert, Robert Caiazzo, Violeta Raverdy, Marie Pigeyre, Abdelilah Arredouani, Jean-Michel Borys, Claire Lévy-Marchal, Jacques Weill, Ronan Roussel, Beverley Balkau, Michel Marre, François Pattou, Thierry Brousseau, Philippe Froguel
BACKGROUND: Salivary (AMY1) and pancreatic (AMY2) amylases hydrolyze starch. Copy number of AMY1A (encoding AMY1) was reported to be higher in populations with a high-starch diet and reduced in obese people. These results based on quantitative PCR have been challenged recently. We aimed to re-assess the relationship between amylase and adiposity using a systems biology approach. METHODS: We assessed the association between plasma enzymatic activity of AMY1 or AMY2, and several metabolic traits in almost 4000 French individuals from D...
February 23, 2017: BMC Medicine
https://www.readbyqxmd.com/read/28218436/differentially-expressed-haptoglobin-as-a-potential-biomarker-for-type-2-diabetic-mellitus-in-hispanic-population
#3
Zhongwei Liu, Du Feng, Danshan Gu, Richard Zheng, Christina Esperat, Weimin Gao
Glycosylated hemoglobin (HbA1c) measurement is currently a primary tool for diagnosis of type 2 diabetes mellitus (T2DM), especially for the assessment of chronic hyperglycemia. However, many studies reported the limitation of using HbA1c for T2DM diagnosis/prognosis, such as poor sensitivities, difficult standardization, and variable cut points across ethnic groups. Therefore, the aim of this study was to discover novel biomarkers associated with elevated HbA1c levels as complementary T2DM diagnostic tools...
February 20, 2017: BioFactors
https://www.readbyqxmd.com/read/28217782/receptor-for-hyaluronan-mediated-motility-rhamm-hmmr-is-a-novel-target-for-promoting-subcutaneous-adipogenesis
#4
S B Bahrami, C Tolg, T Peart, C Symonette, M Veiseh, J U Umoh, D W Holdsworth, J B McCarthy, L G Luyt, M J Bissell, A Yazdani, E A Turley
Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes...
February 20, 2017: Integrative Biology: Quantitative Biosciences From Nano to Macro
https://www.readbyqxmd.com/read/28214757/staining-in-firearm-barrels-after-experimental-contact-shots
#5
C Schyma, K Bauer, J Brünig, C Courts, B Madea
After contact shots to the head biological traces inside firearm barrels can be found. This study was conducted to simulate and to evaluate such staining. Five current handguns of four inch barrel length in the calibre .22 long rifle, 7.65mm Browning, 9mm Luger and .38 special were used to perform 24 contact shots on silicone coated, gelatine filled box models using the triple contrast method. The staining was documented by endoscopy and swabs gathered from both ends of the barrel were analysed by quantitative PCR...
February 10, 2017: Forensic Science International
https://www.readbyqxmd.com/read/28212714/droplet-based-digital-pcr-application-in-cancer-research
#6
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/28205231/analysis-of-ctdna-to-predict-prognosis-and-monitor-treatment-responses-in-metastatic-pancreatic-cancer-patients
#7
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
#8
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/28193149/taqman-probes-as-blocking-agents-for-enriched-pcr-amplification-and-dna-melting-analysis-of-mutant-genes
#9
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
#10
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/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/28183140/correlation-between-pik3ca-mutations-in-cell-free-dna-and-everolimus-efficacy-in-hr-her2-advanced-breast-cancer-results-from-bolero-2
#12
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/28173668/-epidermal-growth-factor-receptor-egfr-mutation-status-before-and-after-acquired-resistance-to-egfr-tyrosine-kinase-inhibitors-in-patients-with-lung-adenocarcinoma
#13
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/28170417/genetic-interaction-mapping-with-microfluidic-based-single-cell-sequencing
#14
John R Haliburton, Wenjun Shao, Adam Deutschbauer, Adam Arkin, Adam R Abate
Genetic interaction mapping is useful for understanding the molecular basis of cellular decision making, but elucidating interactions genome-wide is challenging due to the massive number of gene combinations that must be tested. Here, we demonstrate a simple approach to thoroughly map genetic interactions in bacteria using microfluidic-based single cell sequencing. Using single cell PCR in droplets, we link distinct genetic information into single DNA sequences that can be decoded by next generation sequencing...
2017: PloS One
https://www.readbyqxmd.com/read/28154880/accurate-quantitation-of-circulating-cell-free-mitochondrial-dna-in-plasma-by-droplet-digital-pcr
#15
Wei Ye, Xiaojun Tang, Chu Liu, Chaowei Wen, Wei Li, Jianxin Lyu
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ(0)) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA...
February 2, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28153953/evaluation-of-a-fast-and-fully-automated-platform-to-diagnose-egfr-and-kras-mutations-in-formalin-fixed-and-paraffin-embedded-non-small-cell-lung-cancer-samples-in-less-than-one-day
#16
Laetitia Lambros, Charline Caumont, Briac Guibourg, Fanny Barel, Isabelle Quintin-Roué, Pascale Marcorelles, Jean-Philippe Merlio, Arnaud Uguen
AIMS: Searching for EGFR and KRAS mutations within non-small cell lung carcinoma (NSCLC) samples remains time-consuming and can delay treatment choices in patients with acute deterioration. We evaluated the performances of the fully automated Idylla platform to quickly detect these mutations in NSCLC samples. METHODS: We used the Idylla EGFR Mutation Assay and the Idylla KRAS Mutation Test to analyse 18 formalin-fixed paraffin-embedded NSCLC tumour samples with known EGFR and KRAS mutation status according to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) for EGFRT790M mutations...
February 2, 2017: Journal of Clinical Pathology
https://www.readbyqxmd.com/read/28152506/detection-fidelity-of-ar-mutations-in-plasma-derived-cell-free-dna
#17
Alexa Goldstein, Patricia Valda Toro, Justin Lee, John L Silberstein, Mary Nakazawa, Ian Waters, Karen Cravero, David Chu, Rory L Cochran, Minsoo Kim, Daniel Shinn, Samantha Torquato, Robert M Hughes, Aparna Pallavajjala, Michael A Carducci, Channing J Paller, Samuel R Denmeade, Bruce Kressel, Bruce J Trock, Mario A Eisenberger, Emmanuel S Antonarakis, Ben H Park, Paula J Hurley
Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%...
January 31, 2017: Oncotarget
https://www.readbyqxmd.com/read/28148542/predicting-the-grade-of-dysplasia-of-pancreatic-cystic-neoplasms-using-cyst-fluid-dna-methylation-markers
#18
Tatsuo Hata, Marco Dal Molin, Seung-Mo Hong, Koji Tamura, Masaya Suenaga, Jun Yu, Hiraku Sedogawa, Matthew J Weiss, Christopher L Wolfgang, Anne Marie Lennon, Ralph H Hruban, Michael G Goggins
PURPOSE: Pancreatic cysts are common and pose diagnostic and management challenges. Pancreatic cyst fluid markers have the potential to aid in the management of cysts with concerning imaging findings. Our aim was to evaluate cyst fluid methylated DNA markers for their accuracy for predicting the histologic grade of neoplastic pancreatic cysts. EXPERIMENTAL DESIGN: Pancreatic cyst fluid samples from 183 patients (29 discovery, 154 validation) aspirated after surgical resection were analyzed for methylated DNA at selected genes (SOX17, BNIP3, FOXE1, PTCHD2, SLIT2, EYA4 and SFRP1) using methylation-specific droplet-digital PCR (dd-QMSP)...
February 1, 2017: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
https://www.readbyqxmd.com/read/28147232/use-of-ubiquitous-highly-heterozygous-copy-number-variants-and-digital-droplet-pcr-to-monitor-chimerism-after-allogeneic-haematopoietic-stem-cell-transplantation
#19
John B Whitlam, Ling Ling, Michael Swain, Tom Harrington, Oksana Mirochnik, Ian Brooks, Sara Cronin, Jackie Challis, Vida Petrovic, Damien L Bruno, Francoise Mechinaud, Rachel Conyers, Howard Slater
Chimerism analysis has an important role in the management of allogeneic haematopoietic stem cell transplantation. It informs response to disease relapse, graft rejection and graft-versus-host disease. We have developed a method for chimerism analysis using ubiquitous copy number variation (CNV), which has the benefit of a "negative background" against which multiple independent informative markers are absolutely quantified using digital droplet PCR. A panel of up to 38 CNV markers with homozygous deletion frequencies of approximately 0...
January 29, 2017: Experimental Hematology
https://www.readbyqxmd.com/read/28143479/droplet-based-microfluidic-high-throughput-screening-of-heterologous-enzymes-secreted-by-the-yeast-yarrowia-lipolytica
#20
Thomas Beneyton, Stéphane Thomas, Andrew D Griffiths, Jean-Marc Nicaud, Antoine Drevelle, Tristan Rossignol
BACKGROUND: Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed...
January 31, 2017: Microbial Cell Factories
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