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.
1Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology, Academical Medical Center
In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.
Published April 20, 2015. Keywords: Cellular Biology, Lentivirus, Retrovirus, Organoid, Intestine, Transduction, Stem cell, Lgr5, Endoplasmic reticulum, ER stress, unfolded protein response.
1Department of Physiology and Biophysics, Chicago Medical School
Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.
Published April 20, 2015. Keywords: Cellular Biology, Membrane protein, topology, GFP, fluorescence assay, protease, proteolysis, Digitonin
1Department of Anesthesiology, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College, 3Department of Biochemistry, Weill Cornell Medical College
Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.
Published April 20, 2015. Keywords: Biochemistry, Membrane protein, purification, reconstitution, Poisson statistics, CLC, turnover rate
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