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https://www.readbyqxmd.com/read/27913434/a-study-of-tp53-rna-splicing-illustrates-pitfalls-of-rna-seq-methodology
#1
Sunali Mehta, Peter Tsai, Annette Lasham, Hamish Campbell, Roger Reddel, Antony Braithwaite, Cristin Print
TP53 undergoes multiple RNA-splicing events, resulting in at least nine mRNA transcripts encoding at least 12 functionally different protein isoforms. Antibodies specific to p53 protein isoforms have proven difficult to develop, thus researchers must rely on the transcript information to infer isoform abundance. In this study, we used deep RNA-seq, droplet digital PCR (ddPCR), and real-time quantitative reverse transcriptase PCR (RT-qPCR) from nine human cell lines and RNA-seq data available for tumors in The Cancer Genome Atlas to analyze TP53 splice variant expression...
October 20, 2016: Cancer Research
https://www.readbyqxmd.com/read/27894866/transcriptional-regulation-of-the-sodium-channel-gene-scn5a-by-gata4-in-human-heart
#2
Anna Tarradas, Mel Lina Pinsach-Abuin, Carlos Mackintosh, Oriol Llorà-Batlle, Alexandra Pérez-Serra, Montserrat Batlle, Félix Pérez-Villa, Thomas Zimmer, Ivan Garcia-Bassets, Ramon Brugada, Pedro Beltran-Alvarez, Sara Pagans
Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples...
November 25, 2016: Journal of Molecular and Cellular Cardiology
https://www.readbyqxmd.com/read/27878529/association-between-copy-number-variation-on-metabolic-phenotypes-and-hdl-c-levels-in-patients-with-polycystic-ovary-syndrome
#3
Birgit Knebel, Stefan Lehr, Onno E Janssen, Susanne Hahn, Sylvia Jacob, Ulrike Nitzgen, Dirk Müller-Wieland, Jorg Kotzka
Polygenic diseases with a broad phenotypic spectrum, such as polycystic ovary syndrome (PCOS), present a particular challenge in terms of identifying the underlying genetic mechanisms, nevertheless genetic variants have impact on the individual phenotype. We aimed to determine if next to genetic variations like SNPs further mechanisms might play a role in the pathogenesis of PCOS. We examined the effect of copy-number variations (CNVs) on metabolic phenotypes in PCOS. The intragenic rs1244979, rs2815752 in NEGR1 gene, and rs780094 in GCKR gene were genotyped and CNVs were determined by droplet digital polymerase chain reaction (ddPCR) in PCOS patients (n = 153) and controls without metabolic syndrome (n = 142)...
November 22, 2016: Molecular Biology Reports
https://www.readbyqxmd.com/read/27866520/-research-advancement-on-egfr-mutation-detection-of-cell-free-dna-and-tumor-cell-in-peripheral-blood-of-patients-with-non-small-cell-lung-cancer
#4
Yu Zhang, Yan Xu, Mengzhao Wang
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Epideral growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important treatments currently for advanced NSCLC patients harboring activating EGFR gene mutations, and achieve significant clinical efficacy. T790M mutation occurs in half of NSCLC patents with acquired EGFR-TKI resistance. Screening for EGFR gene mutations in histological and/or circulating tumor cell or DNA samples of NSCLC patients can identify patients who would have a response to EGFR-TKIs or acquire resistance during the treatment...
November 20, 2016: Zhongguo Fei Ai za Zhi, Chinese Journal of Lung Cancer
https://www.readbyqxmd.com/read/27844142/efficiency-and-sensitivity-of-the-digital-droplet-pcr-for-the-quantification-of-antibiotic-resistance-genes-in-soils-and-organic-residues
#5
Laura Cavé, Elisabeth Brothier, Danis Abrouk, Panignimyandé Salomon Bouda, Edmond Hien, Sylvie Nazaret
Droplet digital PCR (ddPCR) allows absolute quantification and tolerance to inhibitors and has been proposed as the method of choice to overcome limitations of qPCR. The aim of this study was to evaluate ddPCR and qPCR performances to detect low copy number and copy number variation of antibiotic resistance genes (sul1 and qnrB genes encoding for resistance to sulfonamides and quinolones, respectively) using bacterial genomic DNA (gDNA) and metagenomic DNA extracted from soil and organic residue samples. With gDNA, qPCR showed a better range of quantification but the lower limit of quantification was at 15 copies of qnrB target vs...
November 14, 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27837339/her2-mrna-transcript-quantitation-in-breast-cancer
#6
K Meehan, B Clynick, B Mirzai, P Maslen, J M Harvey, W N Erber
PURPOSE: The human epidermal growth factor receptor 2 (HER2) status in breast cancer is important for prognostic prediction and the determination of optimal treatment. Current methods rely on protein expression, as determined by immunohistochemistry (IHC), as well as gene amplification as determined by in situ hybridisation (ISH). We explored whether quantitative droplet digital PCR (ddPCR) can be used for the detection and absolute quantitation of HER2 mRNA. METHODS: Digital droplet PCR (ddPCR) was performed for HER2 mRNA on 178 formalin-fixed paraffin-embedded (FFPE) breast cancer specimens...
November 11, 2016: Clinical & Translational Oncology
https://www.readbyqxmd.com/read/27835908/droplet-digital-pcr-analysis-of-notch1-gene-mutations-in-chronic-lymphocytic-leukemia
#7
Angela Minervini, Crescenzio Francesco Minervini, Luisa Anelli, Antonella Zagaria, Paola Casieri, Nicoletta Coccaro, Cosimo Cumbo, Giuseppina Tota, Luciana Impera, Paola Orsini, Claudia Brunetti, Annamaria Giordano, Giorgina Specchia, Francesco Albano
In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time...
November 9, 2016: Oncotarget
https://www.readbyqxmd.com/read/27822863/quantitative-dna-analysis-using-droplet-digital-pcr
#8
Rolf H A M Vossen, Stefan J White
Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27810330/foxl2-402c-g-mutation-can-be-identified-in-the-circulating-tumor-dna-of-patients-with-adult-granulosa-cell-tumors
#9
Anniina Färkkilä, Melissa K McConechy, Winnie Yang, Aline Talhouk, Ying Ng, Amy Lum, Ryan D Morin, Kevin Bushell, Annika Riska, Jessica N McAlpine, C Blake Gilks, Leila Unkila-Kallio, Mikko Anttonen, David G Huntsman
Adult granulosa cell tumors (AGCTs) of the ovary are molecularly characterized by the pathognomonic FOXL2 402C>G (C134W) mutation. To improve diagnostics and follow-up, we developed a specific digital droplet PCR (ddPCR) assay to detect the FOXL2 mutation in the circulating tumor DNA (ctDNA) of AGCT patients. Optimization of the ddPCR assay was performed using a TaqMan primer/probe with the RainDance RainDrop digital PCR system. The ddPCR assay was performed on circulating cell-free DNA extracted from 120 serial plasma samples collected prospectively from 35 AGCT patients...
October 31, 2016: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/27806739/validation-of-droplet-digital-pcr-for-the-detection-and-absolute-quantification-of-borrelia-dna-in-ixodes-scapularis-ticks
#10
Jenny L King, Ashley D Smith, Elizabeth A Mitchell, Michael S Allen
We evaluated the QX200 Droplet Digital PCR (ddPCR™, Bio-Rad) system and protocols for the detection of the tick-borne pathogens Borrelia burgdorferi and Borrelia miyamotoi in Ixodes scapularis nymphs and adults collected from North Truro, Massachusetts. Preliminary screening by nested PCR determined positive infection levels of 60% for B. burgdorferi in these ticks. To investigate the utility of ddPCR as a screening tool and to calculate the absolute number of bacterial genome copies in an infected tick, we adapted previously reported TaqMan®-based qPCR assays for ddPCR...
November 3, 2016: Parasitology
https://www.readbyqxmd.com/read/27802303/uterine-microbiota-and-immune-parameters-associated-with-fever-in-dairy-cows-with-metritis
#11
Soo Jin Jeon, Federico Cunha, Xiaojie Ma, Natalia Martinez, Achilles Vieira-Neto, Rodolfo Daetz, Rodrigo C Bicalho, Svetlana Lima, Jose E P Santos, K Casey Jeong, Klibs N Galvão
OBJECTIVE: This study aimed to evaluate bacterial and host factors causing a fever in cows with metritis. For that, we investigated uterine microbiota using a metagenomic sequencing of the 16S rRNA gene (Study 1), and immune response parameters (Study 2) in metritic cows with and without a fever. PRINCIPAL FINDINGS (STUDY1): Bacterial communities were similar between the MNoFever and MFever groups based on distance metrics of relative abundance of bacteria. Metritic cows showed a greater prevalence of Bacteroidetes, and Bacteroides and Porphyromonas were the largest contributors to that difference...
2016: PloS One
https://www.readbyqxmd.com/read/27800678/precise-quantitation-of-microrna-in-a-single-cell-with-droplet-digital-pcr-based-on-ligation-reaction
#12
Hui Tian, Yuanyuan Sun, Chenghui Liu, Xinrui Duan, Wei Tang, Zhengping Li
MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate single-base difference among miRNAs with simple operations...
November 1, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27793210/investigation-of-ezh2-pathways-for-novel-epigenetic-treatment-strategies-in-oropharyngeal-cancer
#13
Sherif Idris, Cameron Lindsay, Morris Kostiuk, Colin Andrews, David W J Côté, Daniel A O'Connell, Jeffrey Harris, Hadi Seikaly, Vincent L Biron
BACKGROUND: In recent decades, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been rising worldwide as a result of increasing oncogenic human papillomavirus (HPV) infections in the oropharynx. EZH2 is an epigenetic regulatory protein associated with tumor aggressiveness and negative survival outcomes in several human cancers. We aimed to determine the role of EZH2 as a potential therapeutic epigenetic target in HPV-positive and negative OPSCC. METHODS: The expression of EZH2 was measured by immunohistochemistry (IHC) and droplet digital PCR (ddPCR) in 2 HPV-positive and 2 HPV-negative cell lines...
October 28, 2016: Journal of Otolaryngology—Head & Neck Surgery
https://www.readbyqxmd.com/read/27791984/droplet-digital-pcr-is-a-powerful-technique-to-demonstrate-frequent-fgfr1-duplication-in-dysembryoplastic-neuroepithelial-tumors
#14
Frédéric Fina, Doriane Barets, Carole Colin, Corinne Bouvier, Laëtitia Padovani, Isabelle Nanni-Metellus, L'Houcine Ouafik, Didier Scavarda, Andrey Korshunov, David T W Jones, Dominique Figarella-Branger
Dysembryoplastic neuroepithelial tumors (DNT) share V600E mutation in the BRAF gene with other low grade neuroepithelial tumors (LGNTs). FGFR1 internal tandem duplication of the tyrosine-kinase domain (FGFR1-ITD), another genetic alteration that also leads to MAP kinase pathway alteration, has been previously reported in LGNTs by whole-genome sequencing. In the present study we searched for FGFR1-ITD by droplet digital PCR (DDPCR™) and for FGFR1 point mutations by HRM-sequencing in a series of formalin-fixed paraffin-embedded (FFPE) LGNTs including 12 DNT, 2 oligodendrogliomas lacking IDH mutation and 1p/19q co- deletion (pediatric-type oligodendrogliomas; PTOs), 3 pediatric diffuse astrocytomas (PDAs), 14 gangliogliomas (GGs) and 5 pilocytic astrocytomas (PAs)...
October 25, 2016: Oncotarget
https://www.readbyqxmd.com/read/27790140/the-droplet-digital-pcr-a-new-valid-molecular-approach-for-the-assessment-of-b-raf-v600e-mutation-in-hairy-cell-leukemia
#15
Francesca Guerrini, Matteo Paolicchi, Francesco Ghio, Elena Ciabatti, Susanna Grassi, Serena Salehzadeh, Giacomo Ercolano, Maria R Metelli, Marzia Del Re, Lorenzo Iovino, Iacopo Petrini, Giovanni Carulli, Nadia Cecconi, Martina Rousseau, Giulia Cervetti, Sara Galimberti
Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds...
2016: Frontiers in Pharmacology
https://www.readbyqxmd.com/read/27784303/event-specific-detection-of-transgenic-potato-av43-6-g7-using-real-time-and-digital-pcr-methods
#16
Hongwei Gao, Xiaofan Yu, Tingting Deng, Min Sun, Xizhi Xiao, Xin Huang, Ying Chen, Ronggui Li
BACKGROUND: The isolation of unknown DNA sequences flanked by known sequences is an important task in the event-specific detection of GMOs. None of event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic potato AV43-6-G7. RESULTS: The flanking sequence between the exogenous fragment and recombinant chromosome of this potato was successfully acquired through exogenous gene 5'-RACE. The event-specific primers and Taqman probe were designed to amplify fragments spanning the exogenous DNA and potato genomic DNA...
October 27, 2016: BMC Biotechnology
https://www.readbyqxmd.com/read/27770237/egfr-mutation-status-of-paired-cerebrospinal-fluid-and-plasma-samples-in-egfr%C3%A2-mutant-non-small-cell-lung-cancer-with-leptomeningeal-metastases
#17
Jing Zhao, Xin Ye, Yan Xu, Minjiang Chen, Wei Zhong, Yun Sun, Zhenfan Yang, Guanshan Zhu, Yi Gu, Mengzhao Wang
PURPOSE: Central nervous system (CNS) is the prevalent site for metastases in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-relapsed NSCLC patients. To understand the EGFR mutation status in paired cerebrospinal fluid (CSF) and plasma samples after EGFR-TKI treatment failure might be useful to guide the treatment of intra- and extracranial tumors in those patients. METHODS: Paired CSF and plasma samples were collected from seven NSCLC patients with CNS metastases after EGFR-TKI failure...
October 21, 2016: Cancer Chemotherapy and Pharmacology
https://www.readbyqxmd.com/read/27763786/application-of-droplet-digital-pcr-for-estimating-vector-copy-number-states-in-stem-cell-gene-therapy
#18
Huan-Ting Lin, Takashi Okumura, Yukinori Yatsuda, Satoru Ito, Hiromitsu Nakauchi, Makoto Otsu
Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT...
October 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27749322/clinical-validity-of-detecting-circulating-tumor-cells-by-adnatest-assay-compared-with-direct-detection-of-tumor-mrna-in-stabilized-whole-blood-as-a-biomarker-predicting-overall-survival-for-metastatic-castration-resistant-prostate-cancer-patients
#19
Daniel C Danila, Aliaksandra Samoila, Chintan Patel, Nicole Schreiber, Amrita Herkal, Aseem Anand, Diogo Bastos, Glenn Heller, Martin Fleisher, Howard I Scher
Circulating tumor cell (CTC) number measured with the CellSearch assay is prognostic for survival in metastatic castration-resistant prostate cancer before and after therapy. Using a standard operating protocol for sample collection, processing, and analysis, we compared detection rates of CellSearch performed using US Food and Drug Administration-cleared methodology with a second positive selection assay, AdnaTest, and a nonselection polymerase chain reaction (PCR)-based (direct detection PCR [DDPCR]) assay in 55 blood samples from 47 men with progressive metastatic castration-resistant prostate cancer...
September 2016: Cancer Journal
https://www.readbyqxmd.com/read/27733296/the-detection-of-androgen-receptor-splice-variant-7-in-plasma-derived-exosomal-rna-strongly-predicts-resistance-to-hormonal-therapy-in-metastatic-prostate-cancer-patients
#20
Marzia Del Re, Elisa Biasco, Stefania Crucitta, Lisa Derosa, Eleonora Rofi, Cinzia Orlandini, Mario Miccoli, Luca Galli, Alfredo Falcone, Guido W Jenster, Ron H van Schaik, Romano Danesi
BACKGROUND: The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). Due to limitations of the methods available for AR-V7 analysis, the identification of a reliable detection method may facilitate the use of this biomarker in clinical practice. OBJECTIVE: To confirm AR-V7 as a predictor of resistance to hormonal therapy and develop a new approach to assess AR-V7 by highly sensitive digital droplet polymerase chain reaction (ddPCR) in plasma-derived exosomal RNA...
August 26, 2016: European Urology
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