In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH) Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms. Date Published: 10/07/2012, Issue 68; doi: 10.3791/4073 Keywords: Genetics, Issue 68, Infectious Diseases, Immunology, Molecular Biology, nuclei, transcription, var genes, PfEMP1, infected erythrocytes (IE), Plasmodium falciparum, fluorescent in situ hybridization (FISH) Cite this Article Ronander, E., Bengtsson, D.
C., Joergensen, L., Jensen, A. Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. 1. Day 1 Day 2 2. 3. Make a thin blood film by the standard spreading method3. 4. 5. 6. Figure 1. Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip. A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip. Date Published: 9/29/2012, Issue 67; doi: 10.3791/3851 Keywords: Genetics, Issue 67, Molecular Biology, Cellular Biology, RNA, mRNA, Ribonucleoprotein, immunoprecipitation, microarray, PCR, RIP-Chip Cite this Article Dahm, G. M., Gubin, M.
M., Magee, J. D., Techasintana, P., Calaluce, R., Atasoy, U. As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. Experiment preparation Before starting experiment, it is critical to have all reagents, containers and utensils RNase free. 1.
Grow and harvest exponentially growing tissue cells to produce between 2-5 mg of total protein for each RIP. 2. Pre-swell protein A Sepharose (PAS) beads overnight in NT2 buffer (3-4 volumes) with 5% BSA. 3. 4. 5. Figure 1. Figure 2. NIH RO1 A1080870 - To Ulus Atasoy. High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA) The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA). Date Published: 9/13/2012, Issue 67; doi: 10.3791/4415 Keywords: Bioengineering, Issue 67, Genetics, Molecular Biology, Biomedical Engineering, siRNA, transfection, electroporation, microelectrode array, MEA Cite this Article Patel, C., Muthuswamy, J. The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics.
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. 1. 2. 3. 4. 5. Substrate Generation for Endonucleases of CRISPR/Cas Systems. CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length.
Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity. Date Published: 9/08/2012, Issue 67; doi: 10.3791/4277 Keywords: Molecular biology, Issue 67, CRISPR/Cas, endonuclease, in vitro transcription, crRNA, Cas6 Cite this Article Zoephel, J., Dwarakanath, S., Richter, H., Plagens, A., Randau, L. The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. 1. Design PCR primers targeting the spacer regions of a CRISPR cluster. 2. Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons... | JoVE Video. An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described.
The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown. Date Published: 8/17/2012, Issue 66; doi: 10.3791/4141 Keywords: Neuroscience, Issue 66, Physiology, Developmental Biology, cell culture, axon regeneration, axon growth, dorsal root ganglion, spinal cord injury Cite this Article Saijilafu, Zhou, F.
It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. 1. 2. 3. 4. 5. 6. 7. Figure 1. Metrics. An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment... | JoVE Video.
Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging. Date Published: 7/28/2012, Issue 65; doi: 10.3791/4207 Keywords: Cancer Biology, Issue 65, Medicine, Physiology, bladder tumor, orthotopic, bioluminescent, ultrasound, small RNA Cite this Article Kang, M. R., Yang, G., Charisse, K., Epstein-Barash, H., Manoharan, M., Li, L. We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model.
Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco. 1. The human bladder cancer cell line KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) is grown in RPMI-1640 media supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin. 2. 3. 4. 5. saRNA Preparation 6. Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation... | JoVE Video. Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages.
In this article, we describe the method for measurement of microRNAs in microglia. Date Published: 7/23/2012, Issue 65; doi: 10.3791/4097 Keywords: Immunology, Issue 65, Neuroscience, Genetics, microglia, macrophages, microRNA, brain, mouse, real-time PCR, neuroinflammation Veremeyko, T., Starossom, S. Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. 1. Isolation of microglia is performed as was described previously with modifications3. Prepare instruments for dissection and perfusion: scissors, forceps, 10 ml syringe with 27g needle, 15 ml Dounce homogenizer (Teflon/glass), 40 μm nylon cell strainer, 40% and 70% Percoll solutions, 1X PBS. 2. 3. 4. 5. 6. 7.
Figure 1. Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR... | JoVE Video. A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed. Date Published: 7/22/2012, Issue 65; doi: 10.3791/3232 Keywords: Virology, Issue 65, Medicine, Genetics, norovirus, gastroenteritis, RNA extraction, diarrhea, stool samples, PCR, RT-PCR, TaqMan, silica Cite this Article Apaza, S., Espetia, S., Gilman, R. Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen.
Norovirus is second only to rotavirus as the most common cause of diarrhea. 1. Stool samples should be stored frozen to preserve the RNA. 2. 3. 4. 5. 6. 7. DNA Vector-based RNA Interference to Study Gene Function in Cancer. RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo. Date Published: 6/04/2012, Issue 64; doi: 10.3791/4129 Keywords: Cancer Biology, Issue 64, Medicine, Genetics, RNAi, shRNA, gene silencing, mouse xenograft, tumor formation Cite this Article Stovall, D. RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs.
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. 1. 2. 3. 4. 5.
Figure 1. Figure 2. Figure 3. Figure 4. Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells. Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively. Date Published: 6/03/2012, Issue 64; doi: 10.3791/4028 Keywords: Virology, Issue 64, Immunology, Molecular Biology, Influenza A virus, affinity purification, subcellular fractionation, chromatin, vRNP complexes, polymerase Cite this Article Chase, G. Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits.
A schematic flowchart of the protocol is shown in Fig. 1 and a table of reagents is presented below. 1. 2. 3. 4. Detection of Bacteria Using Fluorogenic DNAzymes. We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation.
In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli). Date Published: 5/28/2012, Issue 63; doi: 10.3791/3961 Keywords: Biochemistry, Issue 63, Immunology, Fluorogenic DNAzymes, E. coli, biosensor, bacterial detection Aguirre, S. Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. 1. 2. 3. 4. 5. 6. Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex.
To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth. Date Published: 5/18/2012, Issue 63; doi: 10.3791/3621 Keywords: Neuroscience, Issue 63, electroporation, organotypic slices, RNAi, neurogenesis, neuronal migration, neuronal morphogenesis, brain Cite this Article Lizarraga, S.
B., Coser, K. The cerebral cortex directs higher cognitive functions. We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. 1. 2. Prepare two six-well plates with one culture insert per well using sterile forceps. 3. Prepare RNAi constructs for electroporation. 4. 5. 6. 7. 8. MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR) Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template. Date Published: 5/16/2012, Issue 63; doi: 10.3791/3874 Keywords: Cancer Biology, Issue 63, Medicine, cancer, primer assay, Prostate, microRNA, tumor, qPCR Cite this Article Gordanpour, A., Nam, R. MicroRNAs (miRNAs) are single-stranded, 18–24 nucleotide long, non-coding RNA molecules.
Quantification of miRNA levels in prostate tumor samples. The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. 1. Collect the prostate samples at the time of prostatectomy. 2. Note: Here we have used TRIzol Reagent for extracting RNA, however other kits that isolate small RNA-containing total RNA can also be used. 3. 4. 5. Detection of Viral RNA by Fluorescence in situ Hybridization (FISH) A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques.
This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events. Date Published: 5/05/2012, Issue 63; doi: 10.3791/4002 Keywords: Genetics, Issue 63, Viral genomic RNA, Fluorescence in situ Hybridization, FISH, imaging, genomics Cite this Article Vyboh, K., Ajamian, L., Mouland, A.
Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. Here, we present the method for visual analysis of viral genomic RNA in situ. 1. Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library. The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets.
Here we demonstrate its use and application. Date Published: 4/06/2012, Issue 62; doi: 10.3791/3303 Keywords: Genetics, Issue 62, Target ID, miRNA, ncRNA, RNAi, genomics Cite this Article Coussens, M. J., Forbes, K., Kreader, C., Sago, J., Cupp, C., Swarthout, J. Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library. The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see Figure 4 for plasmid map). 1. 1.
Zeocin is used to select for stably transfected cells. 2. 2. 3. 3. 5. 6. 4. Figure 1. A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation. Chromatin Isolation by RNA Purification (ChIRP) In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection. Chromatin Isolation by RNA Purification (ChIRP) Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans. Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans. RNAi Screening to Identify Postembryonic Phenotypes in C. elegans. In Ovo Electroporation in Embryonic Chick Retina.
Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection. Isolation and Characterization of RNA-Containing Exosomes. In Situ Hybridization for the Precise Localization of Transcripts in Plants. Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling.
Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia. Performing Custom MicroRNA Microarray Experiments. Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA. Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting. RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus. Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography. Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells. RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract. Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays.
Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica. Measuring the Kinetics of mRNA Transcription in Single Living Cells. Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton. High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs. Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved. In vitro Reconstitution of the Active T. castaneum Telomerase. Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation.
Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms. Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery. Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency. Erratum: Visualizing RNA Localization in Xenopus Oocytes. Isolation of Drosophila melanogaster Testes. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. RNAi Interference by dsRNA Injection into Drosophila Embryos. RNAi Interference by dsRNA Injection into Drosophila Embryos. JoVE: Journal of Visualized Experiments - a Video Journal for Biological and Medical Research.