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
1Centre for Infectious Diseases and Microbiology, University of Sydney
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
Published August 6, 2011. Keywords: Molecular Biology, Typing, MRSA, macroarray, molecular epidemiology
JoVE Immunology and Infection
1Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
Published July 22, 2011. Keywords: Immunology, PCR, Salmonella, multiplex, Serovar
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
JoVE Immunology and Infection
1Department of Microbiology, Immunology and Pathology, Colorado State University
Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.
Published July 15, 2011. Keywords: Immunology, Mycobacterium leprae, leprosy, biopsy, STR, VNTR, PCR, fragment length analysis
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
1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.
Published May 22, 2012. Keywords: Basic Protocols, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
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
1School of Biology and the Parker H. Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology
We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.
Published March 19, 2012. Keywords: Genetics, H1 linker histones, histone H1 subtypes, chromatin, RT-PCR, HPLC, gene expression
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