1Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
Published February 13, 2013. Keywords: Genetics, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
1Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine
A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.
Published June 17, 2014. Keywords: Molecular Biology, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
1Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University
Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.
Published January 20, 2014. Keywords: Cellular Biology, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
JoVE Immunology and Infection
1Centre for Antimicrobial Resistance, Alberta Health Services / Calgary Laboratory Services / University of Calgary, 2Department of Pathology & Laboratory Medicine, University of Calgary, 3Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 4Department of Medicine, University of Calgary, 5The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary
We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.
Published September 5, 2013. Keywords: Infection, Microbiology, Genetics, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Bacteria, Bacterial Infections and Mycoses, Life Sciences (General), Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal cassette chromosome mec (SCCmec), SCCmec typing, Multiplex PCR, PCR, sequencing
JoVE Immunology and Infection
1Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, 2Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, 3Department of Pathology, UT Health Science Center at San Antonio, 4Department of Microbiology, UT Health Science Center at San Antonio, 5Cancer Therapy and Research Center, UT Health Science Center at San Antonio
Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.
Published February 24, 2014. Keywords: Infection, In vivo mutagenesis, hematopoietic stem/progenitor cells, LacI mouse model, DNA mutations, E. coli
1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.
Published April 30, 2012. Keywords: Genetics, ChIP, ChIA-PET, Chromatin Interactions, Genomics, Next-Generation Sequencing
1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.
Published August 6, 2012. Keywords: Molecular Biology, Genetics, Cancer Biology, Base excision repair, DNA glycosylase, AP endonuclease, fluorescent, real-time, activity assay, molecular beacon, biomarker, DNA Damage, base lesion
JoVE Clinical and Translational Medicine
1Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston
Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.
Published February 21, 2014. Keywords: Medicine, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
1Department of Neurology and Committee on Neurobiology, The University of Chicago Medical Center, 2Department of Neurology, The University of Chicago Medical Center
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
Published June 13, 2011. Keywords: Neuroscience, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology - Campus North, 2Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus North, 3Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus South
We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.
Published September 10, 2013. Keywords: Developmental Biology, Biochemistry, Vertebrates, Zebrafish, environmental effects (biological and animal), genetics (animal), life sciences, animal biology, animal models, biochemistry, bioengineering (general), Hormones, Hormone Substitutes, and Hormone Antagonists, zebrafish, Danio rerio, chemical screening, luciferase, glucocorticoid, stress, high-throughput screening, receiver operating characteristic curve, in vivo, animal model