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https://www.readbyqxmd.com/read/29156716/using-droplet-digital-pcr-to-analyze-mycn-and-alk-copy-number-in-plasma-from-patients-with-neuroblastoma
#1
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/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
#2
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
#3
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/29151258/clinical-significance-of-disease-specific-myd88-mutations-in-circulating-dna-in-primary-central-nervous-system-lymphoma
#4
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
#5
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/29148297/detection-of-pseudorabies-virus-by-duplex-droplet-digital-pcr-assay
#6
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
#7
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/29141311/-study-on-mosaicism-of-scn1a-gene-mutation-in-parents-of-children-with-dravet-syndrome
#8
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/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
#9
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
#10
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/29115895/droplet-based-digital-pcr-and-next-generation-sequencing-for-monitoring-circulating-tumor-dna-a-cancer-diagnostic-perspective
#11
Mathilde Postel, Alice Roosen, Pierre Laurent-Puig, Valerie Taly, Shu-Fang Wang-Renault
Early detection of cancers through the analysis of ctDNA could have a significant impact on morbidity and mortality of cancer patients. However, using ctDNA for early cancer diagnosis is challenging partly due to the low amount of tumor DNA released in the circulation and its dilution within DNA originating from non-tumor cells. Development of new technologies such as droplet-based digital PCR (ddPCR) or optimized next generation sequencing (NGS) has greatly improved the sensitivity, specificity and precision for the detection of rare sequences...
November 13, 2017: Expert Review of Molecular Diagnostics
https://www.readbyqxmd.com/read/29110175/optimization-and-verification-of-droplet-digital-pcr-even-specific-methods-for-the-quantification-of-gm-maize-das1507-and-nk603
#12
Katarzyna Grelewska-Nowotko, Magdalena Żurawska-Zajfert, Ewelina Żmijewska, Sławomir Sowa
In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform...
November 6, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/29108273/sensitive-droplet-digital-pcr-method-for-detection-of-tert-promoter-mutations-in-cell-free-dna-from-patients-with-metastatic-melanoma
#13
Ashleigh C McEvoy, Leslie Calapre, Michelle R Pereira, Tindaro Giardina, Cleo Robinson, Muhammad A Khattak, Tarek M Meniawy, Antonia L Pritchard, Nicholas K Hayward, Benhur Amanuel, Michael Millward, Melanie Ziman, Elin S Gray
Background: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. Methods: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations...
October 3, 2017: Oncotarget
https://www.readbyqxmd.com/read/29098316/plasma-circular-rna-profiling-of-patients-with-gastric-cancer-and-their-droplet-digital-rt-pcr-detection
#14
Tianwen Li, Yongfu Shao, Liyun Fu, Yi Xie, Linwen Zhu, Weiliang Sun, Rui Yu, Bingxiu Xiao, Junming Guo
To observe the diagnostic values of circular RNAs (circRNAs), their expression profiles between gastric cancer patients' plasma and healthy controls were first assessed by circRNA microarray. Then, circRNA levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR), respectively. A total of 343 differentially expressed circRNAs were found. The top 10 elevated circRNAs in patients were hsa_circ_0088300, hsa_circ_0075825, hsa_circ_0019172, hsa_circ_0000220, hsa_circ_0035277, hsa_circ_0000301, hsa_circ_0000189, hsa_circ_0090080, hsa_circ_0001888, and hsa_circ_0001874...
November 2, 2017: Journal of Molecular Medicine: Official Organ of the "Gesellschaft Deutscher Naturforscher und Ärzte"
https://www.readbyqxmd.com/read/29093617/detection-of-kras-g12d-in-colorectal-cancer-stool-by-droplet-digital-pcr
#15
Susana Olmedillas-López, Dennis César Lévano-Linares, Carmen Laura Aúz Alexandre, Luz Vega-Clemente, Edurne León Sánchez, Alejandro Villagrasa, Jaime Ruíz-Tovar, Mariano García-Arranz, Damián García-Olmo
AIM: To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS: In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation...
October 21, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/29083143/robust-multiplexed-clustering-and-denoising-of-digital-pcr-assays-by-data-gridding
#16
Billy T Lau, Christina Wood-Bouwens, Hanlee P Ji
Digital PCR (dPCR) relies on the analysis of individual partitions to accurately quantify nucleic acid species. The most widely used analysis method requires manual clustering through individual visual inspection. Some automated analysis methods have emerged but do not robustly account for multiplexed targets, low target concentration, and assay noise. In this study, we describe an open source analysis software called Calico that uses "data gridding" to increase the sensitivity of clustering toward small clusters...
November 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29070792/mir-204-is-associated-with-an-endocrine-phenotype-in-human-pancreatic-islets-but-does-not-regulate-the-insulin-mrna-through-mafa
#17
Ilaria Marzinotto, Silvia Pellegrini, Cristina Brigatti, Rita Nano, Raffaella Melzi, Alessia Mercalli, Daniela Liberati, Valeria Sordi, Maurizio Ferrari, Massimo Falconi, Claudio Doglioni, Philippe Ravassard, Lorenzo Piemonti, Vito Lampasona
miR-204 has been proposed to modulate insulin expression in human pancreatic islets by regulating the expression of the MAFA transcript, and in turn insulin transcription. We investigated miR-204 expression in pancreatic endocrine tumors (PET), a panel of human tissues, tissues derived from pancreatic islet purification, and in induced pluripotent stem cells (iPSCs) differentiated towards a pancreatic endocrine phenotype by quantitative real time RT-PCR or droplet digital PCR (ddPCR). In addition, we evaluated the effect of miR-204 up- or down-regulation in purified human islets and in the EndoC-βH1 cell line, as an experimental model of human pancreatic β cells...
October 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29067441/multiplex-picoliter-droplet-digital-pcr-for-quantitative-assessment-of-egfr-mutations-in-circulating-cell-free-dna-derived-from-advanced-non-small-cell-lung-cancer-patients
#18
Qian Yu, Fei Huang, Meilin Zhang, Haiying Ji, Shenchao Wu, Ying Zhao, Chunyan Zhang, Jiong Wu, Beili Wang, Baisheng Pan, Xin Zhang, Wei Guo
To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell‑free DNA (cfDNA) extracted from the plasma of advanced non‑small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) ‑sensitive (19DEL, L858R) and TKI‑resistant (T790 M) mutations...
August 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/29067216/genotyping-tumour-dna-in-cerebrospinal-fluid-and-plasma-of-a-her2-positive-breast-cancer-patient-with-brain-metastases
#19
Giulia Siravegna, Elena Geuna, Benedetta Mussolin, Giovanni Crisafulli, Alice Bartolini, Danilo Galizia, Laura Casorzo, Ivana Sarotto, Maurizio Scaltriti, Anna Sapino, Alberto Bardelli, Filippo Montemurro
BACKGROUND: Central nervous system (CNS) involvement contributes to significant morbidity and mortality in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (mBC) and represents a major challenge for clinicians. Liquid biopsy of cerebrospinal fluid (CSF)-derived circulating tumour DNA (ctDNA) harbours clinically relevant genomic alterations in patients with CNS metastases and could be effective in tracking tumour evolution. METHODS: In a HER2-positive mBC patient with brain metastases, we applied droplet digital PCR (ddPCR) and next-generation whole exome sequencing (WES) analysis to measure ctDNA dynamic changes in CSF and plasma collected during treatment...
2017: ESMO Open
https://www.readbyqxmd.com/read/29051321/liquid-biopsies-using-plasma-exosomal-nucleic-acids-and-plasma-cell-free-dna-compared-with-clinical-outcomes-of-patients-with-advanced-cancers
#20
Lino Möhrmann, Helen Huang, David S Hong, Apostolia M Tsimberidou, Siqing Fu, Sarina Piha-Paul, Vivek Subbiah, Daniel D Karp, Aung Naing, Anne K Krug, Daniel Enderle, Tina Priewasser, Mikkel Noerholm, Erez Eitan, Christine Coticchia, Georg Stoll, Lisa-Marie Jordan, Cathy Eng, Scott Kopetz, Johan Skog, Funda Meric-Bernstam, Filip Janku
PURPOSE: Blood-based liquid biopsies offer easy access to genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. Exosomal nucleic acids (exoNA) originate from living cells, which can better reflect underlying cancer biology. EXPERIMENTAL DESIGN: Next-generation sequencing (NGS) was used to test exosomal nucleic acids (exoNA), and droplet digital PCR (ddPCR) and BEAMing PCR were used to test cfDNA for BRAF V600, KRAS G12/G13, and EGFR exon19del/L858R mutations in 43 patients with progressing advanced cancers...
October 19, 2017: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
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