1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich
Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.
Published August 8, 2013. Keywords: Genetics, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
1Department of Biochemistry, University of Toronto
Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.
Published December 4, 2010. Keywords: Cellular Biology, mRNA nuclear export, microinjection, microscopy, fluorescent in situ hybridization, cell biology
1Virus and Centromere Team, Centre de Génétique et Physiologie Moléculaire et Cellulaire, CNRS UMR 5534, 2Université de Lyon 1, 3Laboratoire d'excellence, LabEX DEVweCAN, 4Institut de Virologie Moléculaire et Structurale, CNRS UPR 3296, 5Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS UMR 5286
We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.
Published January 23, 2014. Keywords: Neuroscience, Life Sciences (General), Virology, Herpes Simplex Virus (HSV), Latency, In situ hybridization, Nuclear organization, Gene expression, Microscopy
1RNA Biology, New England Biolabs
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
Published March 26, 2012. Keywords: Genetics, In vitro transcription, Vaccinia capping enzyme, transfection, T7 RNA Polymerase, RNA synthesis
1School of Life Sciences, Arizona State University, 2Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences
In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.
Published July 25, 2013. Keywords: Neuroscience, Genetics, Behavior, Neurobiology, Molecular Biology, Chemistry, Biochemistry, biology (general), genetics (animal and plant), animal biology, RNA interference, RNAi, double stranded RNA, dsRNA, double gene knockdown, vitellogenin gene, vg, ultraspiracle gene, usp, vitellogenin protein, Vg, ultraspiracle protein, USP, green fluorescence protein, GFP, gustatory perception, proboscis extension response, PER, honey bees, Apis mellifera, animal model, assay
1Biology Department, California State University
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
Published October 16, 2011. Keywords: Developmental Biology, RNA interference, Pristionchus pacificus, microinjection, transgenesis, Caenorhabditis elegans, developmental biology, behavior, gene expression
1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester
This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.
Published December 17, 2013. Keywords: Stem Cell Biology, Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Transcription Factors, General, Gene Therapy, Gene Expression, iPS, OKSM, regenerative medicine
JoVE Immunology and Infection
1Department of Dermatology, University Hospital Zurich
Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.
Published February 2, 2014. Keywords: Immunology, Vaccination, Immunization, intralymphatic immunotherapy, Lymph node injection, vaccines, adjuvants, surgery, anesthesia
JoVE Immunology and Infection
1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 2(CSIC-UCLM-JCCM), Instituto de Investigación en Recursos Cinegéticos IREC
A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.
Published January 20, 2011. Keywords: Infectious Disease, Ticks, RNA interference, genetics, funtional genomics, gene expression, tick-borne pathogens
1Department of Biochemistry, University of Oxford
The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.
Published February 27, 2012. Keywords: Molecular Biology, Drosophila oocytes, wide-field microscopy, cytoskeleton, RNA injection, mRNA localization