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.
1Department of Biochemistry and Molecular Pharmacology, Institute for Systems Genetics, 2Roche Life Science, USA
Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.
Published May 25, 2015. Keywords: Molecular Biology, Sc2.0, synthetic biology, Saccharomyces cerevisiae, genotyping, PCRTag, Cobra, Echo, LightCycler 1536, real time PCR
1School of Life Sciences, Arizona State University, 2Biodesign Institute, Arizona State University
Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.
Published May 25, 2015. Keywords: Molecular Biology, microRNA, luciferase assay, 3' untranslated region, high-throughput, transfection, post-transcriptional gene regulation, cancer
1Department of Ophthalmology, University of Basel, 2Department of Biomedicine, University of Basel
Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.
Published May 22, 2015. Keywords: Molecular Biology, Comet assay analysis, Single-cell gel electrophoresis, DNA breaks, Oxidative stress
1McGovern Institute for Brain Research, Massachusetts Institute of Technology, 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology
Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.
Published May 18, 2015. Keywords: Basic Protocol, Whole blood, catheter, minimally invasive, repeated sampling, plasma, serum, rat, neuroscience, endocrinology, stress
1Department of Neuroscience, University of Minnesota, 2Institute for Translational Neuroscience, University of Minnesota, 3Department of Obstetrics, Gynecology, and Women’s Heath, University of Minnesota, 4Masonic Cancer Center, University of Minnesota
The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.
Published May 10, 2015. Keywords: Cellular Biology, Ubiquitin, Deubiquitinating enzymes, Cellular processes, Pathology, Cancer, Therapeutic targets, Enzymatic activity, Protein degradation.
1Goodman Cancer Research Center, McGill University, 2Department of Biochemistry, McGill University
C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.
Published May 9, 2015. Keywords: Cellular Biology, Oxidative stress, paraquat, Caenorhabditis elegans, reactive oxygen species, organismal death, animal model, nematode
1Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, 2Departamento de Biologia, Centro de Biologia Molecular e Ambiental, Universidade do Minho, 3Department of Neurology and Center for Neuroscience, University of California, Davis Imaging of Dementia and Aging Laboratory, 4Department of Ophthalmology, Institute for Regenerative Cures, University of California, Davis
Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.
Published May 3, 2015. Keywords: Cellular Biology, ion-selective, self-referencing, microelectrode, extracellular ion fluxes, in vivo measurements
1Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health
This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.
Published May 3, 2015. Keywords: Microbiology, Saccharomyces cerevisiae, Kinetochore protein, Ndc10, Ndc80, Sumoylation, Ubiquitination, Post-translational modifications, Protein extracts
1Centre for Education and Research on Ageing & ANZAC Research Institute, University of Sydney and Concord Hospital, 2Ageing and Alzheimers Institute, Concord Hospital, 3Charles Perkins Centre, University of Sydney
The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.
Published April 30, 2015. Keywords: Biophysics, Porosity, Image analysis, Fixation, Perfusion, Image J, Diameter, Fenestrae
1Department of Biology, University of New England
This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.
Published April 30, 2015. Keywords: Behavior, Neuroscience, Drosophila melanogaster, Fruit Flies, Larvae, Life Science, Behavioral Sciences, Locomotion, TriKinetics, Activity, Fly Behavior