JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.
1Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, 2Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney
Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.
Published April 14, 2015. Keywords: Molecular Biology, microRNA, ncRNA, probe-based assays, high-throughput PCR, Nanofluidics / Open Arrays, reverse-transcription, pre-amplification, qPCR
1Department of Biology, San Diego State University
Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.
Published April 14, 2015. Keywords: Molecular Biology, genetic barcoding, fluorescent proteins, retroviral technology, high-throughput, flow cytometry, multiplexing
1Department of Psychiatry and Neurosciences, Faculty of Medicine, Université Laval, 2Centre de recherche de l'Institut universitaire en santé mentale de Québec
Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.
Published April 11, 2015. Keywords: Molecular Biology, Laser capture, microdissection, mRNA, immunolabeling, gene expression, dopamine neurons, RNA sequencing, qRT-PCR
1Department of Developmental Biology, Stanford University School of Medicine
Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.
Published April 8, 2015. Keywords: Cellular Biology, cell cycle, cell biology, systems biology, synchronization, Caulobacter, asymmetric cell division
1Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 2Eck Institute for Global Health, University of Notre Dame, 3Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, 4INRS-Institut Armand-Frappier, 5Department of Biology, Indiana University, 6Department of Biological Sciences, University of Notre Dame
Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.
Published April 7, 2015. Keywords: Microbiology, Surface motility, Swarming, Imaging, Pseudomonas aeruginosa, Salmonella Typhimurium, Bacillus subtilis, Myxococcus xanthus, Flagella
1Department of Biochemistry and Microbiology, University of Victoria
Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.
Published April 5, 2015. Keywords: Microbiology, chemical biology, high-throughput screen, Saccharomyces cerevisiae, yeast knock-out collection, deletion mutant array, chemical genetic interaction, synthetic lethal, synthetic sick, chemogenomics
1Department of Bioengineering, Rice University
Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.
Published March 30, 2015. Keywords: Genetics, recombinase polymerase amplification, isothermal amplification, quantitative, diagnostic, HIV-1, viral load
1Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics
The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.
Published March 30, 2015. Keywords: Cellular Biology, Intracellular compartments, Somatic cell genetics, Na+/H+ exchangers. Intracellular pH measurements. Fast kinetics of ion flux. Kinetic parameters.
1Focal Area Infection Biology, Biozentrum of the University of Basel, 2Proteomics Core Facility, Biozentrum of the University of Basel
The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.
Published March 29, 2015. Keywords: Chemistry, Capture compound, photoactivable crosslinker, mass spectrometry, c-di-GMP effector, EAL, GGDEF, PilZ, Pseudomonas aeruginosa
1Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University
Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.
Published March 25, 2015. Keywords: Molecular Biology, protein delivery, cell-penetrating peptide, zinc-finger, protein transduction domain, chemical biology, molecular biology