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Digital PCR

Hongdo Do, Daniel Cameron, Ramyar Molania, Bibhusal Thapa, Gareth Rivalland, Paul L Mitchell, Carmel Murone, Thomas John, Anthony Papenfuss, Alexander Dobrovic
Identifying circulating tumour DNA (ctDNA) for monitoring of cancer therapy is dependent on the development of readily designed, sensitive cancer-specific DNA markers. Genomic rearrangements that are present in the vast majority of cancers provide such markers.Tumour DNA isolated from two fresh-frozen lung tumours underwent whole genome sequencing. Genomic rearrangements were detected using a new computational algorithm, GRIDSS. Four genomic rearrangements from each tumour were chosen for further study using rearrangement-specific primers...
2016: Advances in Experimental Medicine and Biology
Erina Takai, Yasushi Totoki, Hiromi Nakamura, Mamoru Kato, Tatsuhiro Shibata, Shinichi Yachida
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively...
2016: Advances in Experimental Medicine and Biology
Giovanni Ciancio, Manuela Ferracin, Elena Saccenti, Valentina Bagnari, Ilaria Farina, Federica Furini, Elisa Galuppi, Barbara Zagatti, Francesco Trotta, Massimo Negrini, Marcello Govoni
OBJECTIVES: To evaluate the micro-RNA (miRNA) expression profile in patients with early psoriatic arthritis (PsA) in order to assess the role of miRNAs as potential PsA biomarkers. METHODS: The expression of 723 mature miRNAs in peripheral blood mononuclear cells of early PsA patients in comparison with early-rheumatoid arthritis (ERA) patients and healthy controls (HC) was evaluated using a miRNA microarray. All patients had active disease and were naïve from treatment...
October 7, 2016: Clinical and Experimental Rheumatology
Jingchao Chen, Hongjuan Huang, Shouhui Wei, Zhaofeng Huang, Xu Wang, Chaoxian Zhang
Glyphosate is an important non-selective herbicide that is commonly used worldwide. However, evolved glyphosate-resistant (GR) weeds significantly affect crop yields. Unfortunately, the mechanisms underlying resistance in GR weeds, such as goosegrass (Eleusine indica L.), an annual weed found worldwide, have not been fully elucidated. In this study, transcriptome analysis was conducted to further assess the potential mechanisms of glyphosate resistance in goosegrass. The RNA-seq libraries generated 24,597,462 clean reads...
October 15, 2016: Plant Journal: for Cell and Molecular Biology
David Dobnik, Dejan Štebih, Andrej Blejec, Dany Morisset, Jana Žel
The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR...
October 14, 2016: Scientific Reports
Heather Marion Ames, Ming Yuan, Maria Adelita Vizcaíno, Wayne Yu, Fausto J Rodriguez
Low-grade (WHO I-II) gliomas and glioneuronal tumors represent the most frequent primary tumors of the central nervous system in children. They often have a good prognosis following total resection, however they can create many neurological complications due to mass effect, and may be difficult to resect depending on anatomic location. MicroRNAs have been identified as molecular regulators of protein expression/translation that can repress multiple mRNAs concurrently through base pairing, and have an important role in cancer, including brain tumors...
October 14, 2016: Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc
John F Antiabong, Mafora G Ngoepe, Adakole S Abechi
AIM: The interpretation of conventional polymerase chain reaction (PCR) assay results is often limited to either positive or negative (non-detectable). The more robust quantitative PCR (qPCR) method is mostly reserved for quantitation studies and not a readily accessible technology in laboratories across developing nations. The aim of this study was to evaluate a semi-quantitative method for conventional PCR amplicons using digital image analysis of electrophoretic gel. The potential applications are also discussed...
September 2016: Veterinary World
Philip J Wilson, Stephen L R Ellison
BACKGROUND: Digital PCR (dPCR) is a technique for estimating the concentration of a target nucleic acid by loading a sample into a large number of partitions, amplifying the target and using a fluorescent marker to identify which partitions contain the target. The standard analysis uses only the proportion of partitions containing target to estimate the concentration and depends on the assumption that the initial distribution of molecules in partitions is Poisson. In this paper we describe a way to extend such analysis using the quantification cycle (Cq) data that may also be available, but rather than assuming the Poisson distribution the more general Conway-Maxwell-Poisson distribution is used instead...
October 12, 2016: BMC Bioinformatics
Sebastian Maciak, Katarzyna Michalak, Shiv D Kale, Pawel Michalak
Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F1 hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition...
October 12, 2016: Cytogenetic and Genome Research
Christian D Ahrberg, Andreas Manz, Bong Geun Chung
The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection...
October 5, 2016: Lab on a Chip
E Budisova Svandova, B Vesela, H Lesot, A Poliard, E Matalova
Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8...
October 5, 2016: Histochemistry and Cell Biology
Dean Attali, Roza Bidshahri, Charles Haynes, Jennifer Bryan
Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr - a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface...
2016: F1000Research
Takahiro Kishikawa, Motoyuki Otsuka, Takeshi Yoshikawa, Motoko Ohno, Keisuke Yamamoto, Natsuyo Yamamoto, Ai Kotani, Kazuhiko Koike
Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities...
June 2, 2016: JCI Insight
Ao Huang, Xin Zhang, Shao-Lai Zhou, Ya Cao, Xiao-Wu Huang, Jia Fan, Xin-Rong Yang, Jian Zhou
PURPOSE: Circulating tumor DNA (ctDNA) is increasingly recognized as liquid biopsy to profile tumor genome. Droplet digital PCR (ddPCR) is a highly sensitive and easily operable platform for mutant detection. Here, we tried to detect ctDNA in hepatocellular carcinoma (HCC) patients using ddPCR. METHODS: Studies sequencing the genome of HCCs and COSMIC (Catalogue of Somatic Mutations in Cancer) database were reviewed to identify hotspot mutations. Circulating cell-free DNAs (cfDNAs) extracted from 1 ml preoperative plasma sample were analyzed to detect circulating mutants using ddPCR...
2016: Journal of Cancer
Justine Ellen Marum, Susan Branford
Molecular monitoring plays an essential role in the clinical management of chronic myeloid leukemia (CML) patients, and now guides clinical decision making. Quantitative reverse-transcriptase-polymerase-chain-reaction (qRT-PCR) assessment of BCR-ABL1 transcript levels has become the standard of care protocol in CML. However, further developments are required to assess leukemic burden more efficiently, monitor minimal residual disease (MRD), detect mutations that drive resistance to tyrosine kinase inhibitor (TKI) therapy and identify predictors of response to TKI therapy...
October 2016: Therapeutic Advances in Hematology
Yuta Aizawa, Akihide Koyama, Tomohiko Ishihara, Osamu Onodera, Akihiko Saitoh
BACKGROUND: Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients' clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated. OBJECTIVES: We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)-a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments-increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR...
September 23, 2016: Journal of Clinical Virology: the Official Publication of the Pan American Society for Clinical Virology
Rachel V Purcell, John Pearson, Frank A Frizelle, Jacqueline I Keenan
Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl)...
September 30, 2016: Scientific Reports
Binod Kumar, Salar Khaleghzadegan, Brian Mears, Koji Hatano, Tarana A Kudrolli, Wasim H Chowdhury, David B Yeater, Charles M Ewing, Jun Luo, William B Isaacs, Luigi Marchionni, Shawn E Lupold
The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability...
September 24, 2016: Oncotarget
Abdelghany Hassan Abdelghany, Mohammad A BaSalamah, Shakir Idris, Jawwad Ahmad, Bassem Refaat
BACKGROUND: Vitamin D (VitD) and thymoquinone (TQ) are nutraceutical agents with well-known immunomodulatory and hepatoprotective properties. This study measured whether VitD and TQ, individually or combined, could have direct fibrolytic activities and/or enhanced performance during remedial treatment of liver fibrosis established by CCl4 in rats. METHODS: Eighty five male Wistar rats were used as 10 negative controls (NC) and the remainders were distributed equally into 5 groups: short (S-PC) and long (L-PC) positive controls, TQ, VitD and VitD/TQ groups...
September 29, 2016: Journal of Translational Medicine
D Fumagalli, T R Wilson, R Salgado, X Lu, J Yu, C O'Brien, K Walter, L Y Huw, C Criscitiello, I Laios, V Jose, D N Brown, F Rothé, M Maetens, D Zardavas, P Savas, D Larsimont, M J Piccart-Gebhart, S Michiels, M R Lackner, C Sotiriou, S Loi
BACKGROUND: Estrogen receptor-positive (ER+) breast cancers (BCs) constitute the most frequent BC subtype. The molecular landscape of ER+ relapsed disease is not well characterized. In this study, we aimed to describe the genomic evolution between primary (P) and matched metastatic (M) ER+ BCs after failure of adjuvant therapy. MATERIALS AND METHODS: A total of 182 ER+ metastatic BC patients with long-term follow-up were identified from a single institution. P tumor tissue was available for all patients, with 88 having matched M material...
October 2016: Annals of Oncology: Official Journal of the European Society for Medical Oncology
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