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Digital PCR

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https://www.readbyqxmd.com/read/28645720/direct-circulating-tumor-dna-detection-from-unpurified-plasma-using-a-digital-pcr-platform
#1
David Sefrioui, Ludivine Beaussire, Anne Perdrix, Florian Clatot, Pierre Michel, Thierry Frebourg, Frédéric Di Fiore, Nasrin Sarafan-Vasseur
BACKGROUND: In standard pre-analytical conditions, an isolation step is required for circulating tumor DNA (ctDNA) analysis. The need for this step remains unclear with the development of ultrasensitive detection technologies such as digital PCR (dPCR). The aim of our study was to evaluate the ctDNA detection by dPCR platform either directly from plasma (plasma group, PG) or after an isolation step (isolation group, IG). METHODS: We included 17 patients corresponding to a selection of 43 blood samples in metastatic colorectal cancer patients...
June 20, 2017: Clinical Biochemistry
https://www.readbyqxmd.com/read/28645444/npm1-for-mrd-droplet-like-it-s-hot
#2
Gerald B W Wertheim, Adam Bagg
This commentary highlights the article by Mencia-Trinchant et al that describes a novel digital PCR assay for sensitive detection of minimal residual disease in NPM1 mutated acute myeloid leukemia.
July 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28642342/survival-of-contagious-ovine-digital-dermatitis-codd-associated-treponemes-on-disposable-gloves-after-handling-codd-affected-feet
#3
J W Angell, S R Clegg, D H Grove-White, R W Blowey, S D Carter, J S Duncan, N J Evans
Both contagious ovine digital dermatitis (CODD) and bovine digital dermatitis (BDD) are causes of infectious lameness in sheep and cattle, respectively, and are strongly associated with the presence of specific treponemes, with three different cultivable phylogroups commonly isolated: Treponema medium, Treponema phagedenis and Treponema pedis The aim of this study was to investigate the potential to transmit CODD-associated Treponema species via gloves used when handling visibly clinically affected animals...
June 22, 2017: Veterinary Record
https://www.readbyqxmd.com/read/28640831/ribosomal-dna-copy-number-loss-and-sequence-variation-in-cancer
#4
Baoshan Xu, Hua Li, John M Perry, Vijay Pratap Singh, Jay Unruh, Zulin Yu, Musinu Zakari, William McDowell, Linheng Li, Jennifer L Gerton
Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes...
June 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28637527/rodents-goats-and-dogs-their-potential-roles-in-the-transmission-of-schistosomiasis-in-china
#5
Clare F VAN Dorssen, Catherine A Gordon, Yuesheng Li, Gail M Williams, Yuanyuan Wang, Zhenhua Luo, Geoffrey N Gobert, Hong You, Donald P McManus, Darren J Gray
Schistosomiasis in China has been substantially reduced due to an effective control programme employing various measures including bovine and human chemotherapy, and the removal of bovines from endemic areas. To fulfil elimination targets, it will be necessary to identify other possible reservoir hosts for Schistosoma japonicum and include them in future control efforts. This study determined the infection prevalence of S. japonicum in rodents (0-9·21%), dogs (0-18·37%) and goats (6·9-46·4%) from the Dongting Lake area of Hunan province, using a combination of traditional coproparasitological techniques (miracidial hatching technique and Kato-Katz thick smear technique) and molecular methods [quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR)]...
June 22, 2017: Parasitology
https://www.readbyqxmd.com/read/28636544/analysis-of-esr1-and-pik3ca-mutations-in-plasma-cell-free-dna-from-er-positive-breast-cancer-patients
#6
Takashi Takeshita, Yutaka Yamamoto, Mutsuko Yamamoto-Ibusuki, Mai Tomiguchi, Aiko Sueta, Keiichi Murakami, Yoko Omoto, Hirotaka Iwase
BACKGROUND: The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. METHODS: The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients...
June 14, 2017: Oncotarget
https://www.readbyqxmd.com/read/28629339/droplet-digital-pcr-for-detection-and-quantification-of-circulating-tumor-dna-in-plasma-of-head-and-neck-cancer-patients
#7
Joost H van Ginkel, Manon M H Huibers, Robert J J van Es, Remco de Bree, Stefan M Willems
BACKGROUND: During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). METHODS: TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC)...
June 19, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28626778/single-cell-based-vector-tracing-in-patients-with-ada-scid-treated-with-stem-cell-gene-therapy
#8
Yuka Igarashi, Toru Uchiyama, Tomoko Minegishi, Sirirat Takahashi, Nobuyuki Watanabe, Toshinao Kawai, Masafumi Yamada, Tadashi Ariga, Masafumi Onodera
Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level...
September 15, 2017: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/28626084/optimization-of-ras-braf-mutational-analysis-confirms-improvement-in-patient-selection-for-clinical-benefit-to-anti-egfr-treatment-in-metastatic-colorectal-cancer
#9
Cristina Santos, Daniel Azuara, Rocio Garcia-Carbonero, Pilar García Alfonso, Alfredo Carrato, Elena Elez, Auxiliadora Gómez, Ferran Losa, Clara Montagut, Bartomeu Massuti, Valenti Navarro, Mar Varela, Adriana López-Doriga, Victor Moreno, Manuel Valladares, Jose Luis Manzano, José Maria Viéitez, Enrique Aranda, Xavier Sanjuan, Josep Tabernero, Gabriel Capella, Ramon Salazar
In metastatic colorectal cancer (mCRC) recent studies have shown the importance to accurately quantify low-abundance mutations of RAS pathway because anti-EGFR therapy may depend on certain mutation thresholds. We aimed to evaluate the added predictive value of extended RAS panel testing using two commercial assays and a highly sensitive and quantitative digital PCR (dPCR). Tumor samples from 583 mCRC patients treated with anti-EGFR (n=255) or bevacizumab (n=328) based therapies from several clinical trials and retrospective series from the TTD/RTICC Spanish network were analyzed by cobas®, therascreen® and dPCR...
June 16, 2017: Molecular Cancer Therapeutics
https://www.readbyqxmd.com/read/28625643/sequential-liquid-biopsies-reveal-dynamic-alterations-of-egfr-driver-mutations-and-indicate-egfr-amplification-as-a-new-mechanism-of-resistance-to-osimertinib-in-nsclc
#10
Franciele H Knebel, Fabiana Bettoni, Andrea K Shimada, Manoel Cruz, João Victor Alessi, Marcelo V Negrão, Luiz Fernando L Reis, Artur Katz, Anamaria A Camargo
Osimertinib is an EGFR-T790M-specific TKI, which has demonstrated impressive response rates in NSCLC, after failure to first-line anti-EGFR TKIs. However, acquired resistance to osimertinib is also observed and the molecular mechanisms of resistance are not yet fully understood. Monitoring and managing NSCLC patients who progressed on osimertinib is, therefore, emerging as an important clinical challenge. Sequential liquid biopsies were used to monitor a patient with EGFR-exon19del positive NSCLC, who received erlotinib and progressed through the acquisition of the EGFR-T790M mutation...
June 2017: Lung Cancer: Journal of the International Association for the Study of Lung Cancer
https://www.readbyqxmd.com/read/28625519/multiplex-ultrasensitive-genotyping-of-patients-with-non-small-cell-lung-cancer-for-epidermal-growth-factor-receptor-egfr-mutations-by-means-of-picodroplet-digital-pcr
#11
Masaru Watanabe, Tomoya Kawaguchi, Shun-Ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Osamu Kawashima, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Katsuya Watanabe, Akihide Matsumura, Yasuhiro Koh
Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0...
June 7, 2017: EBioMedicine
https://www.readbyqxmd.com/read/28624953/genome-wide-analysis-of-differential-dna-methylation-in-silver-russell-syndrome
#12
Di Wu, Chunxiu Gong, Chang Su
Silver-Russell Syndrome (SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction, and variable dysmorphic features. Current evidence strongly implicates imprinted genes as an important etiology of SRS. Although almost half of the patients showed DNA hypomethylation at the H19/IGF2 imprinted domain, and approximately 7%-10% of SRS patients have maternal uniparental disomy of chromosome 7 (UPD (7) mat); the rest of the SRS patients shows unknown etiology. In this study, we investigate whether there are further DNA methylation defects in SRS patients...
June 14, 2017: Science China. Life Sciences
https://www.readbyqxmd.com/read/28623522/h3-k27m-mutations-are-extremely-rare-in-posterior-fossa-group-a-ependymoma
#13
Scott Ryall, Miguel Guzman, Samer K Elbabaa, Betty Luu, Stephen C Mack, Michal Zapotocky, Michael D Taylor, Cynthia Hawkins, Vijay Ramaswamy
BACKGROUND: Mutations in the tail of histone H3 (K27M) are frequently found in pediatric midline high-grade glioma's but have rarely been reported in other malignancies. Recently, recurrent somatic nucleotide variants in histone H3 (H3 K27M) have been reported in group A posterior fossa ependymoma (EPN_PFA), an entity previously described to have no recurrent mutations. However, the true incidence of H3 K27M mutations in EPN_PFA is unknown. METHODS: In order to discern the frequency of K27M mutations in histone H3 in EPN_PFA, we analyzed 151 EPN_PFA previously profiled with genome-wide methylation arrays using a validated droplet digital PCR assay...
June 16, 2017: Child's Nervous System: ChNS: Official Journal of the International Society for Pediatric Neurosurgery
https://www.readbyqxmd.com/read/28619728/activated-notch-signaling-cascade-is-correlated-with-stem-cell-differentiation-toward-absorptive-progenitors-after-massive-small-bowel-resection-in-a-rat
#14
Igor Sukhotnik, Arnold G Coran, Yulia Pollak, Eviatar Kuhnreich, Drora Berkowitz, Amulya K Saxena
Notch signaling is thought to act to drive cell versification in the lining of the small intestine. The purpose of the present study was to evaluate the role of Notch signaling pathway in stem cell differentiationin the late stages of intestinal adaptation after massive small bowel resection in a rat. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups of 8 rats each: Sham rats underwent bowel transection and re-anastomosis, SBS- rats underwent 75% small bowel resection. Rats were sacrificed on day 14...
June 15, 2017: American Journal of Physiology. Gastrointestinal and Liver Physiology
https://www.readbyqxmd.com/read/28615715/rnaseq-based-transcriptomics-study-of-smcs-from-carotid-atherosclerotic-plaque-bmp2-and-ids-proteins-are-crucial-regulators-of-plaque-stability
#15
Iraide Alloza, Haize Goikuria, Juan Luis Idro, Juan Carlos Triviño, José María Fernández Velasco, Elena Elizagaray, María García-Barcina, Genoveva Montoya-Murillo, Esther Sarasola, Reyes Vega Manrique, Maria Del Mar Freijo, Koen Vandenbroeck
Carotid artery atherosclerosis is a risk factor to develop cerebrovascular disease. Atheroma plaque can become instable and provoke a cerebrovascular event or else remain stable as asymptomatic type. The exact mechanism involved in plaque destabilization is not known but includes among other events smooth muscle cell (SMC) differentiation. The goal of this study was to perform thorough analysis of gene expression differences in SMCs isolated from carotid symptomatic versus asymptomatic plaques. Comparative transcriptomics analysis of SMCs based on RNAseq technology identified 67 significant differentially expressed genes and 143 significant differentially expressed isoforms in symptomatic SMCs compared with asymptomatic...
June 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28615231/single-nucleotide-polymorphism-leading-to-false-allelic-fraction-by-droplet-digital-pcr
#16
Eric S Christenson, William B Dalton, David Chu, Ian Waters, Karen Cravero, Daniel J Zabransky, Amy DeZern, Ben Ho Park
BACKGROUND: Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS: Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles...
June 14, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/28615230/rapid-rhd-zygosity-determination-using-digital-pcr
#17
Kelly A Sillence, Amr J Halawani, Wajnat A Tounsi, Kirsty A Clarke, Michele Kiernan, Tracey E Madgett, Neil D Avent
BACKGROUND: Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. METHODS: DNA samples extracted from 58 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)(2) to determine gene dosage by digital PCR...
June 14, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/28614272/quantification-of-microrna-21-and-microrna-125b-in-melanoma-tissue
#18
Anne Wandler, Rikke Riber-Hansen, Henrik Hager, Stephen J Hamilton-Dutoit, Henrik Schmidt, Boye S Nielsen, Magnus Stougaard, Torben Steiniche
Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours...
June 13, 2017: Melanoma Research
https://www.readbyqxmd.com/read/28611392/targeted-sequencing-of-both-dna-strands-barcoded-and-captured-individually-by-rna-probes-to-identify-genome-wide-ultra-rare-mutations
#19
Qing Wang, Xu Wang, Pheobe S Tang, Grace M O'leary, Ming Zhang
Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq)...
June 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28602964/droplet-digital-pcr-improved-the-egfr-mutation-diagnosis-with-pleural-fluid-samples-in-non-small-cell-lung-cancer-patients
#20
Xiaoyan Li, Yi Liu, Weiwei Shi, Huayan Xu, Haixu Hu, Zhengwei Dong, Guanshan Zhu, Yun Sun, Bing Liu, Hongjun Gao, Chuanhao Tang, Xiaoqing Liu
BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of nucleic acid. However, its application has not been reported in pleural fluid, which is an ideal sample source for epidermal growth factor receptor (EGFR) mutation analysis in non-small-cell lung cancer (NSCLC) patients. METHODS: The extracted DNA from supernatants of pleural fluid was selected from our sample bank and re-analyzed by our previously established ddPCR assay...
June 8, 2017: Clinica Chimica Acta; International Journal of Clinical Chemistry
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