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https://www.readbyqxmd.com/read/28548029/digital-assays-part-ii-digital-protein-and-cell-assays
#1
Amar S Basu
A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays...
May 1, 2017: SLAS Technology
https://www.readbyqxmd.com/read/28546538/droplet-digital-pcr-versus-qpcr-for-gene-expression-analysis-with-low-abundant-targets-from-variable-nonsense-to-publication-quality-data
#2
Sean C Taylor, Genevieve Laperriere, Hugo Germain
Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower...
May 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28542711/preventing-relapse-after-haematopoietic-stem-cell-transplantation-for-acute-leukaemia-the-role-of-post-transplantation-minimal-residual-disease-mrd-monitoring-and-mrd-directed-intervention
#3
REVIEW
Xiao-Dong Mo, Meng Lv, Xiao-Jun Huang
Relapse is the main cause of treatment failure after allogeneic haematopoietic stem cell transplantation (allo-HSCT) for acute leukaemia (AL). Post-transplantation minimal residual disease (MRD) monitoring enables risk stratification and identifies AL patients at higher risk of relapse. MRD assessment primarily involves the determination of leukaemia-associated immunophenotypic patterns using multiparameter flow cytometry, and the polymerase chain reaction (PCR)-based evaluation of expression levels of leukaemia-related genes (specific reciprocal gene rearrangements and other mutation types)...
May 23, 2017: British Journal of Haematology
https://www.readbyqxmd.com/read/28542573/development-of-real-time-pcr-and-loop-mediated-isothermal-amplification-lamp-assays-for-the-differential-detection-of-digital-dermatitis-associated-treponemes
#4
Kelly Anklam, Megan Kulow, Wataru Yamazaki, Dörte Döpfer
Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T...
2017: PloS One
https://www.readbyqxmd.com/read/28540249/liquid-biopsies-for-bladder-cancer
#5
EDITORIAL
Douglas G Ward, Richard T Bryan
The development of accurate urinary biomarkers for the non-invasive detection of urothelial bladder cancer (UBC) could transform patient pathways by reducing reliance on cystoscopy, and the identification of highly prognostic (or even predictive) biomarkers could better guide patient management. A number of approaches are being utilised to address these challenges in both urinary- and plasma-borne tumour DNA (tDNA), so-called "liquid biopsies". Next generation sequencing (NGS) and droplet digital PCR (ddPCR) allow detection of very low levels of such tDNA amongst a large excess of non-tumour DNA, the former permitting large mutation panels to be assessed and the latter potentially identifying ultrarare mutant alleles yet restricted for multiplexing...
April 2017: Translational Andrology and Urology
https://www.readbyqxmd.com/read/28536327/-clinical-significance-of-circulating-tumor-dna-and-its-future-prospects
#6
Shigeyoshi Harada
"Liquid biopsy"is a diagnostic medium to detect very small amount of tumor-derived molecules in blood for selecting precise clinical treatments and giving a diagnosis. This article focuses on circulating tumor DNA(ctDNA)and describes its features, clinical significance as well as future prospects with recent findings in this field. "Liquid biopsy"using ctDNAis facilitated by recent developments of highly sensitive technologies, including digital PCR. "BEAMing"is a digital PCR method that can detect rare mutations in ctDNAand will be introduced with some clinical evidences in this article...
May 2017: Gan to Kagaku Ryoho. Cancer & Chemotherapy
https://www.readbyqxmd.com/read/28527094/circulating-tumor-dna-measurement-by-picoliter-droplet-based-digital-pcr-and-vemurafenib-plasma-concentrations-in-patients-with-advanced-braf-mutated-melanoma
#7
Fanny Garlan, Benoit Blanchet, Nora Kramkimel, Alicja Puszkiel, Jean-Louis Golmard, Gaelle Noe, Nicolas Dupin, Pierre Laurent-Puig, Michel Vidal, Valerie Taly, Audrey Thomas-Schoemann
BACKGROUND: Circulating tumor DNA (ctDNA) has been reported as a prognostic marker in melanoma. In BRAF V600-mutant melanoma, a plasma under-exposure to vemurafenib could favor emerging resistance but no biological data are available to support this hypothesis. OBJECTIVE: We aimed to investigate the relationship between vemurafenib plasma concentrations and the ctDNA plasma concentration during follow-up of BRAF-mutated melanoma patients. PATIENTS AND METHODS: Eleven patients treated with single-agent vemurafenib for advanced BRAF V600-mutant melanoma were analyzed in an exploratory monocentric study...
May 19, 2017: Targeted Oncology
https://www.readbyqxmd.com/read/28525852/absolute-quantification-of-dna-methylation-using-microfluidic-chip-based-digital-pcr
#8
Zhenhua Wu, Yanan Bai, Zule Cheng, Fangming Liu, Ping Wang, Dawei Yang, Gang Li, Qinghui Jin, Hongju Mao, Jianlong Zhao
Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact...
May 12, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28525762/minimal-residual-disease-monitoring-of-acute-myeloid-leukemia-by-massively-multiplex-digital-pcr-in-patients-with-npm1-mutations
#9
Nuria Mencia-Trinchant, Yang Hu, Maria Antonina Alas, Fatima Ali, Bas J Wouters, Sangmin Lee, Ellen K Ritchie, Pinkal Desai, Monica L Guzman, Gail J Roboz, Duane C Hassane
The presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. However, detection is complicated by the existence of hundreds of potential frameshift insertions, clonal heterogeneity, and absence of sequence information when the NPM1 mutation is identified using capillary electrophoresis...
May 16, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28522907/droplet-digital-pcr-for-quantification-of-itga6-in-a-stool-mrna-assay-for-the-detection-of-colorectal-cancers
#10
Elizabeth Herring, Shigeru Kanaoka, Éric Tremblay, Jean-François Beaulieu
AIM: To investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening. METHODS: ddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods...
April 28, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28520748/inflammation-related-microrna-expression-level-in-the-bovine-milk-is-affected-by-mastitis
#11
Yu-Chang Lai, Takuro Fujikawa, Tadashi Maemura, Takaaki Ando, Go Kitahara, Yasuyuki Endo, Osamu Yamato, Masateru Koiwa, Chikara Kubota, Naoki Miura
MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System...
2017: PloS One
https://www.readbyqxmd.com/read/28508913/digitally-encoded-silica-microparticles-for-multiplexed-nucleic-acid-detection
#12
Weiwei Xu, Chao Chen, Xiaodong Ma, Lihua Yuan, Shenquan Liu, Kexiao Zheng, Jiong Li
A robust high throughput suspension array was proposed to analyze biomolecules. To exploit its capacity for nucleic acid detection, we designed a multiplex nested asymmetric PCR (MNAS PCR) that can produce single stranded DNA (ssDNA) efficiently without complicated optimization. Multiplexed HPV genotyping was demonstrated with high selectivity, high sensitivity and rapid hybridization within 20 minutes.
May 16, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28507181/fli1-deficiency-induces-cxcl6-expression-in-dermal-fibroblasts-and-endothelial-cells-contributing-to-the-development-of-fibrosis-and-vasculopathy-in-systemic-sclerosis
#13
Takashi Taniguchi, Yoshihide Asano, Kouki Nakamura, Takashi Yamashita, Ryosuke Saigusa, Yohei Ichimura, Takehiro Takahashi, Tetsuo Toyama, Ayumi Yoshizaki, Shinichi Sato
OBJECTIVE: CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels. METHODS: mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively...
May 15, 2017: Journal of Rheumatology
https://www.readbyqxmd.com/read/28502728/technical-validation-of-a-next-generation-sequencing-assay-for-detecting-clinically-relevant-levels-of-breast-cancer-related-single-nucleotide-variants-and-copy-number-variants-using-simulated-cell-free-dna
#14
Xin Yang, Yuxing Chu, Rui Zhang, Yanxi Han, Lucheng Zhang, Yu Fu, Dan Li, Rongxue Peng, Dongdong Li, Jiansheng Ding, Ziyang Li, Meiru Zhao, Kuo Zhang, Tian Lu, Lang Yi, Qisheng Wu, Guigao Lin, Jiehong Xie, Tao Liu, Ling Yang, Xin Yi, Jinming Li
Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28499695/dapagliflozin-modulates-glucagon-secretion-in-an-sglt2-independent-manner-in-murine-alpha-cells
#15
A Solini, G Sebastiani, L Nigi, E Santini, C Rossi, F Dotta
AIM: SGLT2 inhibitors reduce renal glucose uptake through an insulin-independent mechanism. They also increase glucagon concentration, although the extent to which this is due to a direct effect on pancreatic alpha cells remains unclear. METHODS: In the present work, αTC1 cells treated with the SGLT2 inhibitor dapagliflozin (Dapa) were analyzed for glucose transporters, molecular mediators of hormone secretion, glucagon and GLP-1 release, and the effects of somatostatin...
May 9, 2017: Diabetes & Metabolism
https://www.readbyqxmd.com/read/28499138/human-pegivirus-1-in-the-csf-of-patients-with-hiv-associated-neurocognitive-disorder-hand-may-be-derived-from-blood-in-highly-viraemic-patients
#16
Diana Hardie, Heidi Smuts
BACKGROUND: Human pegivirus-1 (HPgV-1) infection in the brain has not been extensively examined and its association with disease remains unconfirmed. In a high throughput sequencing study to look for infectious agents that could play a role in HIV-associated neurocognitive disorder (HAND), this virus was detected in 3 of 8 CSF samples. OBJECTIVES: To determine the significance of this finding, additional patients were screened and the viral load and viral diversity in blood and CSF were examined...
April 6, 2017: Journal of Clinical Virology: the Official Publication of the Pan American Society for Clinical Virology
https://www.readbyqxmd.com/read/28497651/harnessing-the-power-of-digital-droplet-pcr-to-conduct-real-world-microbial-competitions
#17
Helen A Murphy
The budding yeast, Saccharomyces cerevisiae, has a long and storied history as a model organism for genetic, cellular and molecular biological research. More recently, researchers have sought to understand the ecology and evolution of its sister species, Saccharomyces paradoxus, in part to put our vast knowledge of the model yeast into its natural context (Replansky et al. ). However, the research tools have been limited, and most investigations into natural populations have either been descriptions of patterns of biogeography or taken the organism back into the laboratory for mating, growth and competition assays (Kuehne et al...
May 2017: Molecular Ecology Resources
https://www.readbyqxmd.com/read/28490568/noninvasive-detection-of-f8-int22h-related-inversions-and-sequence-variants-in-maternal-plasma-of-hemophilia-carriers
#18
Irena Hudecova, Peiyong Jiang, Joanna Davies, Y M Dennis Lo, Rezan A Kadir, Rossa W K Chiu
Direct detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among hemophilia patients, namely those involving int22h-related inversions disrupting the F8 gene, poses additional challenges due to its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia...
May 10, 2017: Blood
https://www.readbyqxmd.com/read/28489996/cancer-associated-mutations-in-endometriosis-without-cancer
#19
Michael S Anglesio, Nickolas Papadopoulos, Ayse Ayhan, Tayyebeh M Nazeran, Michaël Noë, Hugo M Horlings, Amy Lum, Siân Jones, Janine Senz, Tamer Seckin, Julie Ho, Ren-Chin Wu, Vivian Lac, Hiroshi Ogawa, Basile Tessier-Cloutier, Rami Alhassan, Amy Wang, Yuxuan Wang, Joshua D Cohen, Fontayne Wong, Adnan Hasanovic, Natasha Orr, Ming Zhang, Maria Popoli, Wyatt McMahon, Laura D Wood, Austin Mattox, Catherine Allaire, James Segars, Christina Williams, Cristian Tomasetti, Niki Boyd, Kenneth W Kinzler, C Blake Gilks, Luis Diaz, Tian-Li Wang, Bert Vogelstein, Paul J Yong, David G Huntsman, Ie-Ming Shih
BACKGROUND: Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis. METHODS: We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients)...
May 11, 2017: New England Journal of Medicine
https://www.readbyqxmd.com/read/28487685/effects-of-inhibitors-on-the-transcriptional-profiling-of-gluconobater-oxydans-nl71-genes-after-biooxidation-of-xylose-into-xylonate
#20
Yuanyuan Miao, Yi Shen, Yong Xu
D-Xylonic acid belongs to the top 30 biomass-based platform chemicals and represents a promising application of xylose. Until today, Gluconobacter oxydans NL71 is the most efficient microbe capable of fermenting xylose into xylonate. However, its growth is seriously inhibited when concentrated lignocellulosic hydrolysates are used as substrates due to the presence of various degraded compounds formed during biomass pretreatment. Three critical lignocellulosic inhibitors were thereby identified, i.e., formic acid, furfural, and 4-hydroxybenzaldehyde...
2017: Frontiers in Microbiology
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