JoVE General
Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics
Molekulare Pflanzenphysiologie, Max-Planck-Institut
Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.
JoVE General
A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid
1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 2Department of Rheumatology, Tufts Medical Center
A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.
JoVE Neuroscience
Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury
Department of Anesthesiology, University of Texas Medical Branch
We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.
JoVE General
Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform
School of Health Sciences, Purdue University
Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.
JoVE General
Microarray Analysis for Saccharomyces cerevisiae
Vermont Genetics Network, The University of Vermont
In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.
JoVE General
Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes
1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington
Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.
JoVE General
Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana
Crop and Soil Sciences, Cornell University
This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.
JoVE General
Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors
Institute for Biological Interfaces, Forschungszentrum Karlsruhe
We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.
JoVE General
Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences
Department of Cell and Developmental Biology, University of Pennsylvania
We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.
JoVE General
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Wellcome Trust Centre for Human Genetics, University of Oxford
Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.
JoVE General
Expression Analysis of Mammalian Linker-histone Subtypes
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.
JoVE General
Processing the Loblolly Pine PtGen2 cDNA Microarray
1Warnell School of Forestry and Natural Resources, University of Georgia (UGA), 2Instituto de Biologia Experimental e Tecnológica, Instituto Tecnologia Química e Biológica UNL, Av. da República
The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.
JoVE Immunology and Infection
Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry
Institute for Molecular Infection Biology (IMIB), University of Würzburg
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
JoVE Neuroscience
Transcriptome Analysis of Single Cells
1Department of Pharmacology, University of Pennsylvania, 2The Penn Genome Frontiers Institute, University of Pennsylvania
In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
JoVE General
Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues
1Department of Horticulture and Crop Science, The Ohio State University, 2Department of Plant Pathology, North Carolina State University
We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.
JoVE General
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
1Molecular Microbiology and Immunology, University of Missouri, 2Department of Surgery, University of Missouri, 3Child Health, University of Missouri
A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.
JoVE General
Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines
Department of Surgery, Weill Cornell Medical College
A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.
JoVE General
Generation of Single-Cell Suspensions from Mouse Neural Tissue
Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.
JoVE Neuroscience
An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells
Department of Neuroscience, University of Minnesota
This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.
JoVE Clinical and Translational Medicine
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
JoVE Neuroscience
A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain
1Division of Neurotoxicology, National Center for Toxicological Research, 2Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, 3Office of Planning, Finance, and Information Technology, National Center for Toxicological Research
This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.
JoVE Clinical and Translational Medicine
In vivo Dual Substrate Bioluminescent Imaging
Case Comprehensive Cancer Center, Case Western Reserve University
Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.
JoVE General
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Children'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.
JoVE General
Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration
Department of Biological Sciences, University of Notre Dame
Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.
JoVE Immunology and Infection
Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky
We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.
JoVE Neuroscience
Assaying β-amyloid Toxicity using a Transgenic C. elegans Model
1Institute for Behavioral Genetics, University of Colorado, 2Integrative Physiology, University of Colorado
The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ). Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.
JoVE Clinical and Translational Medicine
Skeletal Muscle Gender Dimorphism from Proteomics
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
JoVE Bioengineering
Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages
1Stem Cell Lab, School of Psychiatry, Faculty of Medicine, The University of New South Wales, 2Siriraj Center of Excellence for Stem cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 3Neuropsychiatric Institute, Prince of Wales Hospital
We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.
JoVE General
Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR
Department of Biological Sciences, Southwestern Oklahoma State University
Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.
JoVE General
Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana
1Department of Energy - Plant Research Laboratory, Michigan State University (MSU), 2Department of Biochemistry and Molecular Biology, Michigan State University (MSU)
The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.
JoVE General
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
JoVE General
Microdissection of Zebrafish Embryonic Eye Tissues
Department of Biological Sciences, Purdue University
This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.
JoVE General
Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos
Department of Genetics, University of Georgia
This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.
JoVE General
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
Gene Expression Division, Bio-Rad Laboratories
This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.
JoVE General
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
1Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University, 2Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences
We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.
JoVE Clinical and Translational Medicine
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Stem Cell and Cancer Research Institute, McMaster University
The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
JoVE Clinical and Translational Medicine
Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury
Department of Biology, Unit of Zoology, University of Fribourg, Fribourg, Switzerland
Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.
JoVE General
Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR
Department of Entomology, The Ohio State University
Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.
JoVE General
A Quantitative Assay for Insulin-expressing Colony-forming Progenitors
1Department of Biotechnology & Bioinformatics, California State University Channel Islands, 2Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, 3The Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope
A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.
JoVE Neuroscience
Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina
Department of Neurobiology, University of Oldenburg
This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.
JoVE Immunology and Infection
Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, 2Department of Chemistry, University of Nebraska-Lincoln
The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.
JoVE General
In Situ Hybridization for the Precise Localization of Transcripts in Plants
The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.
JoVE General
Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models
Basic Medical Sciences, University of Arizona College of Medicine - Phoenix
A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.
JoVE General
Bacterial Gene Expression Analysis Using Microarrays
Department of Environmental Toxicology, University of California Santa Cruz - UCSC
JoVE General
RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis
School of Health Sciences, Purdue University
The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.
JoVE Clinical and Translational Medicine
Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
1Cancer Biology Department, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Department of Pharmaceutical Sciences, University of Hawaii at Hilo College of Pharmacy
Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse.
JoVE General
Single Oocyte Bisulfite Mutagenesis
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
JoVE General
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
JoVE Immunology and Infection
Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine
Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.
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