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https://www.readbyqxmd.com/read/29156792/liquid-biopsy-in-colon-cancer-comparison-of-different-circulating-dna-extraction-systems-following-absolute-quantification-of-kras-mutations-using-intplex-allele-specific-pcr
#1
Vera Kloten, Nadine Rüchel, Nadina Ortiz Brüchle, Janina Gasthaus, Nils Freudenmacher, Florian Steib, Jolein Mijnes, Julian Eschenbruch, Marcel Binnebösel, Ruth Knüchel, Edgar Dahl
Non-invasive molecular analysis of circulating tumor DNA (ctDNA) is a promising application in personalized cancer management, although there is still much to learn about the biological characteristics of ctDNA. The present study compared absolute amounts of KRAS mutated ctDNA and total circulating cell-free DNA (cfDNA) in colorectal cancer (CRC) patients (n=50) from various stages and healthy controls (n=8) by Intplex allele-specific and digital droplet PCR. In addition, the impact of two prominent extraction techniques (silica-based membrane vs...
October 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29156777/patients-with-nsclc-may-display-a-low-ratio-of-p-t790m-vs-activating-egfr-mutations-in-plasma-at-disease-progression-implications-for-personalised-treatment
#2
Marzia Del Re, Paola Bordi, Iacopo Petrini, Eleonora Rofi, Francesca Mazzoni, Lorenzo Belluomini, Enrico Vasile, Giuliana Restante, Francesco Di Costanzo, Alfredo Falcone, Antonio Frassoldati, Ron H N van Schaik, Christi M J Steendam, Antonio Chella, Marcello Tiseo, Riccardo Morganti, Romano Danesi
Introduction: NSCLC harboring activating mutations of EGFR is highly sensitive to first-line EGFR-tyrosine kinase inhibitors (TKIs), but drug resistance depending on the EGFR mutation p.T790M will occur in about 50-60% of patients. Detailed information on the amount of p.T790M plasmatic level associated with resistance to EGFR-TKIs and guidance to treatment with p.T790M-effective TKI depending on these levels, is lacking. Methods: This study enrolled p.T790M-positive patients (n=49) affected by EGFR-mutated NSCLC at progression to first-line EGFR-TKIs and, in selected cases (n=5), after second-line treatment with osimertinib...
October 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29156716/using-droplet-digital-pcr-to-analyze-mycn-and-alk-copy-number-in-plasma-from-patients-with-neuroblastoma
#3
Marco Lodrini, Annika Sprüssel, Kathy Astrahantseff, Daniela Tiburtius, Robert Konschak, Holger N Lode, Matthias Fischer, Ulrich Keilholz, Angelika Eggert, Hedwig E Deubzer
The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients...
October 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29156604/takamatsu-h-comparison-of-minimal-residual-disease-detection-by-multiparameter-flow-cytometry-aso-qpcr-droplet-digital-pcr-and-deep-sequencing-in-patients-with-multiple-myeloma-who-underwent-autologous-stem-cell-transplantation-j-clin-med-2017-6-91
#4
https://www.readbyqxmd.com/read/29152135/evaluation-of-pre-analytical-conditions-and-comparison-of-the-performance-of-several-digital-pcr-assays-for-the-detection-of-major-egfr-mutations-in-circulating-dna-from-non-small-cell-lung-cancers-the-circan_0-study
#5
Jessica Garcia, Eric Dusserre, Valérie Cheynet, Pierre Paul Bringuier, Karen Brengle-Pesce, Anne-Sophie Wozny, Claire Rodriguez-Lafrasse, Gilles Freyer, Marie Brevet, Léa Payen, Sébastien Couraud
Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay...
October 20, 2017: Oncotarget
https://www.readbyqxmd.com/read/29152132/high-baalc-copy-numbers-in-peripheral-blood-prior-to-allogeneic-transplantation-predict-early-relapse-in-acute-myeloid-leukemia-patients
#6
Madlen Jentzsch, Marius Bill, Juliane Grimm, Julia Schulz, Karoline Goldmann, Stefanie Beinicke, Janine Häntschel, Wolfram Pönisch, Georg-Nikolaus Franke, Vladan Vucinic, Gerhard Behre, Thoralf Lange, Dietger Niederwieser, Sebastian Schwind
High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes...
October 20, 2017: Oncotarget
https://www.readbyqxmd.com/read/29152076/profiling-tumour-heterogeneity-through-circulating-tumour-dna-in-patients-with-pancreatic-cancer
#7
Patricia Adamo, Caroline M Cowley, Christopher P Neal, Vilas Mistry, Karen Page, Ashley R Dennison, John Isherwood, Robert Hastings, JinLi Luo, David A Moore, Pringle J Howard, Martins L Miguel, Catrin Pritchard, Margaret Manson, Jacqui A Shaw
The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed late so that surgery is rarely curative. Earlier detection could significantly increase the likelihood of successful treatment and improve survival. The aim of the study was to provide proof of principle that point mutations in key cancer genes can be identified by sequencing circulating free DNA (cfDNA) and that this could be used to detect early PDACs and potentially, premalignant lesions, to help target early effective treatment. Targeted next generation sequencing (tNGS) analysis of mutation hotspots in 50 cancer genes was conducted in 26 patients with PDAC, 14 patients with chronic pancreatitis (CP) and 12 healthy controls with KRAS status validated by digital droplet PCR...
October 20, 2017: Oncotarget
https://www.readbyqxmd.com/read/29151258/clinical-significance-of-disease-specific-myd88-mutations-in-circulating-dna-in-primary-central-nervous-system-lymphoma
#8
Keiichiro Hattori, Mamiko Sakata-Yanagimoto, Yasuhito Suehara, Yasuhisa Yokoyama, Takayasu Kato, Naoki Kurita, Hidekazu Nishikii, Naoshi Obara, Shingo Takano, Eiichi Ishikawa, Akira Matsumura, Yuichi Hasegawa, Shigeru Chiba
Recent sequencing studies demonstrated the MYD88 L265P mutation in more than 70% of primary central nervous system lymphomas (PCNSL), and the clinical significance of this mutation has been proposed as diagnostic and prognostic markers in PCNSL. On the other hand, mutational analyses using cell-free DNAs have been reported in a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P mutation can be identified in cell-free DNA from PCNSL patients, we performed droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in consecutive 14 PCNSL patients from whom paired tumor-derived DNA and cell-free DNA was available at diagnosis...
November 19, 2017: Cancer Science
https://www.readbyqxmd.com/read/29150911/jak2-v617f-mutation-can-be-reliably-detected-in-serum-using-droplet-digital-pcr
#9
C F Nystrand, W Ghanima, A Waage, C M Jonassen
INTRODUCTION: Detection of the JAK2 V617F mutation is a key step in the diagnosis of myeloproliferative neoplasms (MPN). Sensitive real-time quantitative PCR (qPCR) detection on peripheral blood (PB) is the most widely used method. The main objective of this study was to determine whether serum, the most common material available in archival biobanks, is a good liquid biopsy for detecting and quantifying the JAK2 V617F mutation using droplet digital PCR (ddPCR). METHODS: Paired PB and serum samples from 66 patients with MPN were used...
November 17, 2017: International Journal of Laboratory Hematology
https://www.readbyqxmd.com/read/29148539/extrauterine-high-grade-serous-carcinomas-with-bilateral-adnexal-involvement-as-the-only-two-disease-sites-are-clonal-based-on-tp53-sequencing-results-implications-for-biology-classification-and-staging
#10
Naveena Singh, Asma Faruqi, Friedrich Kommoss, W Glenn McCluggage, Giorgia Trevisan, Janine Senz, Amy Lum, C Blake Gilks, Michael Anglesio
A previous multicenter study of 67 cases of Stage I/II tubo-ovarian high-grade serous carcinoma with complete tubal sampling identified 7 cases in which there were only two disease sites, comprising tumor involving opposite adnexa with no extra-adnexal involvement. This study aimed to determine whether such low-stage extrauterine high-grade serous carcinomas with only two sites of involvement, located on opposite adnexa, have identical or different TP53 mutations in order to investigate their clonal relationship...
November 17, 2017: Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc
https://www.readbyqxmd.com/read/29148297/detection-of-pseudorabies-virus-by-duplex-droplet-digital-pcr-assay
#11
Meishen Ren, Hua Lin, Shijie Chen, Miao Yang, Wei An, Yin Wang, Changhua Xue, Yinjie Sun, Yubao Yan, Juan Hu
Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines...
November 1, 2017: Journal of Veterinary Diagnostic Investigation
https://www.readbyqxmd.com/read/29145448/steps-to-achieve-quantitative-measurements-of-microrna-using-two-step-droplet-digital-pcr
#12
Erica V Stein, David L Duewer, Natalia Farkas, Erica L Romsos, Lili Wang, Kenneth D Cole
Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies...
2017: PloS One
https://www.readbyqxmd.com/read/29142202/genomic-mosaicism-in-paternal-sperm-and-multiple-parental-tissues-in-a-dravet-syndrome-cohort
#13
Xiaoxu Yang, Aijie Liu, Xiaojing Xu, Xiaoling Yang, Qi Zeng, Adam Yongxin Ye, Zhe Yu, Sheng Wang, August Yue Huang, Xiru Wu, Qixi Wu, Liping Wei, Yuehua Zhang
Genomic mosaicism in parental gametes and peripheral tissues is an important consideration for genetic counseling. We studied a Chinese cohort affected by a severe epileptic disorder, Dravet syndrome (DS). There were 56 fathers who donated semen and 15 parents who donated multiple peripheral tissue samples. We used an ultra-sensitive quantification method, micro-droplet digital PCR (mDDPCR), to detect parental mosaicism of the proband's pathogenic mutation in SCN1A, the causal gene of DS in 112 families. Ten of the 56 paternal sperm samples were found to exhibit mosaicism of the proband's mutations, with mutant allelic fractions (MAFs) ranging from 0...
November 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29142136/size-composition-and-evolution-of-hiv-dna-populations-during-early-antiretroviral-therapy-and-intensification-with-maraviroc
#14
Antoine Chaillon, Sara Gianella, Steven M Lada, Josué Perez-Santiago, Parris Jordan, Caroline Ignacio, Maile Karris, Douglas Richman, Sanjay R Mehta, Susan J Little, Joel O Wertheim, Davey M Smith
Residual viremia is common during antiretroviral therapy (ART), and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART Intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial, and followed for a median of 107 weeks. Participants started ART with (n=9) or without (n=9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR...
November 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/29141311/-study-on-mosaicism-of-scn1a-gene-mutation-in-parents-of-children-with-dravet-syndrome
#15
A J Liu, X X Yang, X J Xu, Q X Wu, X J Tian, X L Yang, X R Wu, L P Wei, Y H Zhang
Objective: To investigate the clinical phenotypes and the mutant allele proportion of parents with SCN1A gene mutation mosaicism of Dravet syndrome (DS) children, thus to provide guidance for family reproduction and prenatal diagnosis. Method: The clinical data and peripheral blood DNA samples of DS patients with a SCN1A gene mutation proved by Sanger sequencing were collected prospectively from February 2005 to November 2016 in Department of Pediatrics, Peking University First Hospital. The same mutation was searched in parents and other available relatives...
November 2, 2017: Zhonghua Er Ke za Zhi. Chinese Journal of Pediatrics
https://www.readbyqxmd.com/read/29137608/the-pennycress-thlaspi-arvense-l-nectary-structural-and-transcriptomic-characterization
#16
Jason B Thomas, Marshall E Hampton, Kevin M Dorn, M David Marks, Clay J Carter
BACKGROUND: Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. RESULTS: Pennycress flowers contain four equivalent nectaries located extrastaminally at the base of the insertion sites of short and long stamens...
November 14, 2017: BMC Plant Biology
https://www.readbyqxmd.com/read/29136689/-detection-of-epidermal-growth-factor-receptor-gene-mutations-in-different-types-of-non-small-cell-lung-cancer-by-droplet-digital-pcr-and-amplification-refractory-mutation-system
#17
R Li, S B Ye, Y He, X Wang, N Wu, Q Y Xia, Q Shen, S S Shi
Objective: To compare amplification refractory mutation system(ARMS) and droplet digital PCR (ddPCR) in the detection of epidermal growth factor receptor (EGFR) gene mutations in patients with non-small cell lung cancer (NSCLC), and to investigate the clinical value of ddPCR. Methods: A total of 79 specimens of NSCLC, including 22 cases of cell block, 18 cases of surgical specimens, 12 cases of biopsy specimens and 27 cases of plasma samples, were analyzed for the mutation status of EGFR gene by ARMS and droplet digital PCR method...
November 8, 2017: Zhonghua Bing Li Xue za Zhi Chinese Journal of Pathology
https://www.readbyqxmd.com/read/29122835/multiple-hotspot-mutations-scanning-by-single-droplet-digital-pcr
#18
Charles Decraene, Amanda Silveira, François-Clément Bidard, Audrey Vallee, Marc Michel, Samia Melaabi, Anne Vincent-Salomon, Adrien Saliou, Alexandre Houy, Maud Milder, Olivier Lantz, Marc Ychou, Marc G Denis, Jean-Yves Pierga, Marc-Henri Stern, Charlotte Proudhon
BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR)(12), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS() exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes...
November 9, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/29120463/compartmentalized-partnered-replication-for-the-directed-evolution-of-genetic-parts-and-circuits
#19
Zhanar Abil, Jared W Ellefson, Jimmy D Gollihar, Ella Watkins, Andrew D Ellington
Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29118738/monitoring-of-dual-crispr-cas9-mediated-steroidogenic-acute-regulatory-protein-gene-deletion-and-cholesterol-accumulation-using-high-resolution-fluorescence-in-situ-hybridization-in-a-single-cell
#20
Jinwoo Lee, Colin Jefcoate
Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria...
2017: Frontiers in Endocrinology
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