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droplet pcr

Cosimo Cumbo, Luciana Impera, Crescenzio Francesco Minervini, Paola Orsini, Luisa Anelli, Antonella Zagaria, Nicoletta Coccaro, Giuseppina Tota, Angela Minervini, Paola Casieri, Claudia Brunetti, Antonella Russo Rossi, Elisa Parciante, Giorgina Specchia, Francesco Albano
For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology...
February 16, 2018: Oncotarget
Meenakshi Mehrotra, Rajesh R Singh, Sanam Loghavi, Dzifa Yawa Duose, Bedia A Barkoh, Carmen Behrens, Keyur P Patel, Mark J Routbort, Scott Kopetz, Russell R Broaddus, L Jeffrey Medeiros, Ignacio I Wistuba, Rajyalakshmi Luthra
A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton...
February 13, 2018: Oncotarget
Benedikt G Brink, Justin Meskas, Ryan R Brinkman
Motivation: Droplet digital PCR (ddPCR) is an emerging technology for quantifying DNA. By partitioning the target DNA into ∼20000 droplets, each serving as its own PCR reaction compartment, a very high sensitivity of DNA quantification can be achieved. However, manual analysis of the data is time consuming and algorithms for automated analysis of non-orthogonal, multiplexed ddPCR data are unavailable, presenting a major bottleneck for the advancement of ddPCR transitioning from low-throughput to high- throughput...
March 9, 2018: Bioinformatics
Laura Lupini, Anna Moretti, Cristian Bassi, Alessio Schirone, Massimo Pedriali, Patrizia Querzoli, Roberta Roncarati, Antonio Frassoldati, Massimo Negrini
Approximately 70% of breast cancers (BCs) express estrogen receptor alpha (ERα) and are treated with endocrine therapy. However, the effectiveness of this therapy is limited by innate or acquired resistance in approximately one-third of patients. Activating mutations in the ESR1 gene that encodes ERα promote critical resistance mechanisms. Here, we developed a high sensitivity approach based on enhanced-ice-COLD-PCR for detecting ESR1 mutations. The method produced an enrichment up to 100-fold and allowed the unambiguous detection of ESR1 mutations even when they consisted of only 0...
March 12, 2018: Scientific Reports
Nicoletta Coccaro, Antonella Zagaria, Paola Orsini, Luisa Anelli, Giuseppina Tota, Paola Casieri, Luciana Impera, Angela Minervini, Crescenzio F Minervini, Cosimo Cumbo, Elisa Parciante, Anna Mestice, Mario Delia, Claudia Brunetti, Giorgina Specchia, Francesco Albano
Most Acute Promyelocytic Leukemia (APL) patients express PML-RARA fusion; in rare cases RARA is rearranged with partner genes other than PML. To date, only two patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an Acute Myeloid Leukemia (AML) patient with morphology resembling APL without involvement of the RARA gene. Molecular and Fluorescent In Situ Hybridization (FISH) analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci...
March 9, 2018: Human Pathology
C B Thomsen, T F Hansen, R F Andersen, J Lindebjerg, L H Jensen, A Jakobsen
BACKGROUND: Precision medicine calls for an early indicator of treatment efficiency. Circulating tumor DNA (ctDNA) is a promising marker in this setting. Our prospective study explored the association between disease development and change of ctDNA during first line chemotherapy in patients with RAS/RAF mutated metastatic colorectal cancer (mCRC). METHODS: The study included 138 patients with mCRC receiving standard first line treatment. In patients with RAS/RAF mutated tumor DNA the same mutation was quantified in the plasma using droplet digital PCR...
March 12, 2018: Journal of Experimental & Clinical Cancer Research: CR
Lin Li, Meilin He, Hang Xiao, Xiaoqian Liu, Kai Wang, Yuanshu Zhang
BACKGROUND/AIMS: Acetic acid (AcOH), a short-chain fatty acid, is reported to have some beneficial effects on metabolism. Therefore, the aim of this study was to investigate the regulatory mechanism of acetic acid on hepatic lipid metabolism in BRL-3A cells. METHODS: We cultured and treated BRL-3A cells with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPKα inhibitor). The total lipid droplet area was measured by oil red O staining, and the triglyceride content was determined by a triglyceride detection kit...
March 6, 2018: Cellular Physiology and Biochemistry
Liesbeth Van Wesenbeeck, Leen Janssens, Hanne Meeuws, Ole Lagatie, Lieven Stuyver
Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples...
March 12, 2018: Epigenetics: Official Journal of the DNA Methylation Society
Kathryn B Manheimer, Nihir Patel, Felix Richter, Joshua Gorham, Angela C Tai, Jason Homsy, Marko T Boskovski, Michael Parfenov, Elizabeth Goldmuntz, Wendy K Chung, Martina Brueckner, Martin Tristani-Firouzi, Deepak Srivastava, Jonathan G Seidman, Christine E Seidman, Bruce D Gelb, Andrew J Sharp
Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs)...
March 11, 2018: Human Mutation
Wei-Neng Feng, Wei-Quan Gu, Ning Zhao, Ying-Ming Pan, Wei Luo, Hua Zhang, Jian-Miao Liang, Jie Yang, Yan-Ming Deng
BACKGROUND: Liquid biopsy is emerging as an important approach for tumor genotyping in non-small cell lung cancer, ddPCR and SuperARMS are both methods with high sensitivity and specificity for detecting EGFR mutation in plasma. We aimed to compare ddPCR and SuperARMS to detect plasma EGFR status in a cohort of advanced NSCLC patients. METHOD: A total of 79 tumor tissues and paired plasma samples were collected. The EGFR mutation status in tissue was tested by ADx-ARMS, matched plasma was detected by ddPCR and SuperARMS, respectively...
March 8, 2018: Translational Oncology
Banjun Ruan, Zheng Zhu, Zhao Yan, Wei Yang, Dongsheng Zhai, Li Wang, Zichen Ye, Huanyu Lu, An Xiang, Jingwei Liang, Yinghao Jiang, Chengming Xu, Zhenyu Wang, Ming Wei, Xiaoying Lei, Xiaorui Cao, Zifan Lu
BACKGROUND: The reciprocal fate decision of mesenchymal stem cells (MSCs) to either bone or adipocytes is determined by Wnt-related signaling and the glucagon-like peptide-1 receptor (GLP-1R). Azoramide, an ER stress alleviator, was reported to have an antidiabetic effect. In this study, we investigated the function of azoramide in regulating the lineage determination of MSCs for either adipogenic or osteogenic differentiation. METHODS: In this study, microcomputed tomography and histological analysis on bone morphogenetic protein (BMP)2-induced parietal periosteum bone formation assays, C3H10T1/2 and mouse bone marrow MSC-derived bone formation and adipogenesis assays, and specific staining for bone tissue and lipid droplets were used to evaluate the role of azoramide on the lineage determination of MSC differentiation...
March 9, 2018: Stem Cell Research & Therapy
Francesco Martelli, Jessica Mencarini, Arianna Rocca, Nunzia Della Malva, Dario Bartolozzi, Simone Giannecchini
Current evidence suggests that Polyomavirus (PyV) microRNAs (miRNAs) circulating in biological fluids may be relevant to understanding viral persistence. Here, the expression of polyomavirus BKPyV, JCPyV, MCPyV and SV40 miRNAs in saliva was investigated to evaluate PyV prevalence/persistence in the oral cavity. PyV-DNA status and PyV-miRNA expression was examined in paired saliva and plasma samples of 100 HIV-infected patients and of 50 healthy subjects using digital droplet PCR and PyV-miRNA-5p stem-loop RT-PCR...
March 5, 2018: Virus Research
Marzia Del Re, Riccardo Marconcini, Giulia Pasquini, Eleonora Rofi, Caterina Vivaldi, Francesco Bloise, Giuliana Restante, Elena Arrigoni, Chiara Caparello, Maria Grazia Bianco, Stefania Crucitta, Iacopo Petrini, Enrico Vasile, Alfredo Falcone, Romano Danesi
BACKGROUND: PD-L1 expression in tumour tissues is widely used to select patients to receive anti-PD-1/PD-L1 antibodies, but data are lacking on the correlation of plasma PD-L1 levels with the effect of treatments. METHODS: To investigate the association between PD-L1 mRNA in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients with melanoma (n=18) and NSCLC (n=8), blood was obtained at time point 0 and after 2 months. Exosomal PD-L1 mRNA was measured by digital droplet PCR...
March 6, 2018: British Journal of Cancer
Alessandra Ruggiero, Alessandro Cozzi-Lepri, Apostolos Beloukas, Douglas Richman, Saye Khoo, Andrew Phillips, Anna Maria Geretti
Background: Persistence of plasma HIV-1 RNA during seemingly effective antiretroviral thereapy (ART) is incompletely understood. Using an ultrasensitive assay, this cross-sectional study investigated residual plasma HIV-1 RNA in subjects maintained on firstline ART with continuous viral load suppression <50 copies/mL for ≤15 years without recognized viral load blips or treatment interruptions and explored its relationship with the duration of suppressive ART, efavirenz concentrations in plasma, 2-LTR circular HIV-1 DNA (2-LTRc DNA) in peripheral blood mononuclear cells, and cellular (CD4 plus CD26/CD38/CD69; CD8 plus CD38/HLA-DR/DP/DQ) and soluble (sCD14, sCD27, sCD30, IL-6) markers of immune activation in peripheral blood...
February 2018: Open Forum Infectious Diseases
S Robinson, M Follo, D Haenel, M Mauler, D Stallmann, M Tewari, D Duerschmied, K Peter, C Bode, I Ahrens, M Hortmann
BACKGROUND: micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. AIMS: We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). METHODS: A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR...
April 15, 2018: International Journal of Cardiology
Frida A Zink, Luke R Tembrock, Alicia E Timm, Todd M Gilligan
The silver Y moth [Autographa gamma (Linneaus) (Noctuidae: Plusiinae)] is a pervasive crop pest in its native range but has not been found in moth surveys in the United States. Specimens of A. gamma are often intercepted at U.S. ports of entry, so the risk of introduction of this invasive species is high. Currently, identification of Plusiinae adults captured in domestic surveys is done by morphlogical comparison; however, this method is time consuming and misidentifications have occurred in the past. A recent study outlined a real-time PCR assay capable of rapidly identifying individual A...
March 1, 2018: Journal of Economic Entomology
Anna Buder, Maximilian J Hochmair, Sophia Schwab, Tatjana Bundalo, Peter Schenk, Peter Errhalt, Romana E Mikes, Gudrun Absenger, Kurt Patocka, Bernhard Baumgartner, Ulrike Setinek, Otto C Burghuber, Helmut Prosch, Robert Pirker, Martin Filipits
INTRODUCTION: Osimertinib is standard treatment for patients with advanced EGFR T790M-mutated NSCLC who have been pre-treated with EGFR-TKIs. We studied whether cell-free plasma DNA for T790M detection can be used to select patients for osimertinib treatment in clinical routine. METHODS: From April 2015 to November 2016, we included 119 patients with advanced EGFR-mutated NSCLC who had progressed under treatment with an EGFR-TKI. The T790M mutation status was assessed in cell-free plasma DNA by droplet digital PCR (ddPCR) in all patients and by tissue analyses in selected patients...
March 2, 2018: Journal of Thoracic Oncology
Joshua A Steele, A Denene Blackwood, John F Griffith, Rachel T Noble, Kenneth C Schiff
Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult...
March 1, 2018: Water Research
Ling Wei, Li Xie, Xingwu Wang, Hongxin Ma, Liyan Lv, Lisheng Liu, Xianrang Song
Genotype-directed targeted therapy has become one of the standard treatment options for non-small cell lung cancer (NSCLC). There have been numerous limitations associated with mutation analysis of tissue samples. Consequently, mutational profile analysis of circulating cell-free DNA (cfDNA) by highly sensitive droplet digital PCR (ddPCR) assay has been developed. Possibly due to differences in cfDNA concentrations, previous studies have shown numerous discrepancies in mutation detection consistency between tissue and cfDNA...
March 1, 2018: Laboratory Investigation; a Journal of Technical Methods and Pathology
Ryo Ariyasu, Shingo Nishikawa, Ken Uchibori, Tomoko Oh-Hara, Takahiro Yoshizawa, Yosuke Dotsu, Junji Koyama, Masafumi Saiki, Tomoaki Sonoda, Satoru Kitazono, Noriko Yanagitani, Atsushi Horiike, Naohiko Inase, Kazuo Kasahara, Makoto Nishio, Ryohei Katayama
OBJECTIVES: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that can overcome resistance due to the Thr790Met (T790M) mutation. However, osimertinib occasionally shows limited efficacy in a small population of patients. We investigated the correlation between the ratio of T790M to EGFR activating mutation and the response to osimertinib. MATERIALS AND METHODS: Between April 2016 and April 2017, 44 patients started osimertinib therapy at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research...
March 2018: Lung Cancer: Journal of the International Association for the Study of Lung Cancer
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