Name: sample preparation and analysis of rnaseq-based gene expression data from zebrafish
Uploaded: 2017-10-27T12:30:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish at JoVE.com

Diese Arbeit wurde unterstützt durch R01DK102001 (N.A.Z.), P30DK072488 (N.A.Z.) und T32DK098107 (T.L.H. und J.E.N).

<ol>
	<li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish. , (2002).</li><li>Detrich, H. W., Zon, L., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Disease Models and Chemical Screens. , (2017).</li><li>Detrich, H. W., Zon, L. I., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Genetics, Genomics, and Transcriptomics. , (2016).</li><li>Detrich, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Genetics, Genomics and Informatics. , (2011).</li><li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Hwang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+editing+in+zebrafish+using+a+CRISPR-Cas+system.">Efficient genome editing in zebrafish using a CRISPR-Cas system.</a> Nat Biotechnol. 31 (3), 227-229 (2013).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Rossi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+compensation+induced+by+deleterious+mutations+but+not+gene+knockdowns.">Genetic compensation induced by deleterious mutations but not gene knockdowns.</a> Nature. 524 (7564), 230-233 (2015).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Samsa, L. A., Fleming, N., Magness, S., Qian, L., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Single+Cells+from+Zebrafish+Embryos.">Isolation and Characterization of Single Cells from Zebrafish Embryos.</a> J Vis Exp. (109), (2016).</li><li>. IDT Primerquest Tool Available from: <a target="_blank" href="https://www.idtdna.com/Primerquest/Home/Index">https://www.idtdna.com/Primerquest/Home/Index</a> (2017)</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6 (10), 986-994 (1996).</li><li>Leitch, C. C., Lodh, S., Prieto-Echague, V., Badano, J. L., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+body+proteins+regulate+Notch+signaling+through+endosomal+trafficking.">Basal body proteins regulate Notch signaling through endosomal trafficking.</a> J Cell Sci. 127 (Pt 11), 2407-2419 (2014).</li><li>Lodh, S., Hostelley, T. L., Leitch, C. C., O'Hare, E. A., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+on+beta-cell+mass+by+disruption+of+Bardet-Biedl+syndrome+or+Alstrom+syndrome+genes.">Differential effects on beta-cell mass by disruption of Bardet-Biedl syndrome or Alstrom syndrome genes.</a> Hum Mol Genet. 25 (1), 57-68 (2016).</li><li>Hostelley, T. L., Lodh, S., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+organism+transcriptome+analysis+of+zebrafish+models+of+Bardet-Biedl+Syndrome+and+Alstrom+Syndrome+provides+mechanistic+insight+into+shared+and+divergent+phenotypes.">Whole organism transcriptome analysis of zebrafish models of Bardet-Biedl Syndrome and Alstrom Syndrome provides mechanistic insight into shared and divergent phenotypes.</a> BMC Genomics. 17, 318 (2016).</li></ol>

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In diesem Protokoll beschriebene Ansatz bietet eine relativ schnelle und kostengünstige Strategie für Transkriptom-Level-Analyse der ganze Tiere oder bestimmte sortierte Zellpopulationen. Der Zebrabärbling bietet eine vorteilhafte Modell für diese Art der Studie wegen der Leichtigkeit und Schnelligkeit beim generieren großer Mengen an Material, die einfache Implementierung von genetischen oder experimentelle Umweltbedingungen und die Verfügbarkeit eines großen ab Spektrum an transgenen Reporter Linien zur Isolatio...

Vergleichende Analyse der globalen Genexpression ist ein wertvolles Werkzeug, neuartige Gene zur beobachteten Phänotypen zu identifizieren. Solche Analysen in der Regel verlassen sich auf quantitative Bewertung der Abschrift Fülle im Vergleich zwischen experimentellen und Kontrollproben. Gezielte Ansätze, wie z. B. qRT-PCR sind relativ schnelle und genaue Untersuchung der Veränderungen der einzelnen Genexpression. RNA-Sequenzierung (RNASeq) bietet einen breiten, Hypothese-freie Ansatz um wesentliche Änderungen in der Genexpression zwischen Proben, so dass es nun den Standard für solche Untersuchungen über experimentelle Systeme zu identifizieren.
Zebrafisch entstanden als prominente Modell in vielen Bereichen der Krankheit. Ursprünglich entwickelt für ihre Nützlichkeit in Entwicklungsbiologie Studien aufgrund ihrer hohen Fruchtbarkeit und relativ geringen Kosten für Wartung, experimentellen Einsatz von Zebrafisch entwickelt hat, um umfassen eine Vielzahl von Phänotypen aus embryonalen bis adulten Stadien als auch als ein Vielzahl von molekularen Tests1,2,3. In der Tat, diese Vorteile machen die molekularen mechanistische Studien schnelle und kostengünstige wegen der Leichtigkeit des Erwerbs von großen Mengen an Material kombiniert mit der Leichtigkeit des genetischen und umweltbedingten Manipulation in allen Phasen des Lebens. Darüber hinaus die transparente Art der Zebrafisch-Embryonen und Larven machen es ideal für die Erzeugung von Zell- und gewebespezifische transgenen Reporter Linien in Vivo Visualisierung von diskreten Zelle Bevölkerungen4ermöglicht. Ausbeutung von solchen Linien ermöglicht globale gen Expressionsanalyse in bestimmten isolierten Zelle Arten basierend auf Reporter-gen-Expression.
Hier präsentieren wir Ihnen ein umfassendes Protokoll für globale Genanalyse Ausdruck mit RNASeq nach der Kultur der Zebrafisch-Embryonen. Genetischen experimentelle Manipulationen, einschließlich Morpholino (MO)-aufgrund vorübergehender gen-Knockdown oder CRISPR-vermittelten Genom-Bearbeitung, wurden präsentiert an anderer Stelle5,6,7. Wir daher konzentrieren sich auf ein detailliertes Protokoll zur Isolierung von RNA aus ganzen Embryonen oder transgene Reporter exprimierenden Zellen, gefolgt von einfachen numerischen Analyse der RNASeq Ergebnisse mit Weg-Tools und gen Ontology (gehen Sie) Begriffe sortiert. Zu guter Letzt haben wir eine Strategie für die Validierung von Veränderungen der Genexpression durch quantitative Reverse Transkriptase PCR (qRT-PCR) aufgenommen. Diese Protokolle sind für Zebrafisch-Embryonen, die eine Vielzahl von experimentellen Bedingungen, auch im Vergleich der genetische Mutanten oder Umweltbedingungen ausgesetzt.

<table><tbody><tr><td>&lt;strong&gt;Commercial Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TriZol</td><td>Thermo Scientific</td><td>15596026</td><td>lysis reagent</td></tr><tr><td>TrypLE</td><td>Gibco</td><td>12604013</td><td>dissociation buffer 1</td></tr><tr><td>FACSMax</td><td>Genlantis</td><td>T200100</td><td>dissociation buffer 2</td></tr><tr><td>DEPC-treated water</td><td>Sigma</td><td>95284</td><td></td></tr><tr><td>FirstStrand cDNA conversion</td><td>Thermo Scientific</td><td>K1621</td><td>cDNA conversion kit</td></tr><tr><td>2X SYBR Green Master Mix</td><td>Roche</td><td>4707516001</td><td>qRT-PCR Master Mix</td></tr><tr><td>FACS buffer</td><td>Fisher Scientific</td><td>50-105-9042</td><td></td></tr><tr><td>chloroform</td><td>Sigma Aldrich</td><td>288306</td><td></td></tr><tr><td>sodium acetate</td><td>Sigma Aldrich</td><td>S2889</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Zebrafish Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tuebingen</td><td>ZIRC</td><td>ZL57</td><td></td></tr><tr><td>ins2a:mCherry</td><td>ZIRC</td><td>ZL1483</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>40 micron cell strainer</td><td>Sigma</td><td>CLS431750</td><td></td></tr><tr><td>FACS tube</td><td>BD Falcon</td><td>352063</td><td></td></tr><tr><td>hemocytometer</td><td>Sigma</td><td>Z359629</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Inverted Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Illumina HiSeq</td><td>Illumina</td><td></td><td></td></tr><tr><td>LightCycler 480</td><td>Roche</td><td></td><td></td></tr><tr><td>Mating tanks 1.0L Crossing Tank Set</td><td>Aquaneering</td><td>ZHCT100</td><td></td></tr><tr><td>FACS tube 5 mL polypropylene tube</td><td>BD Falcon</td><td>352063</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Consensus Path DB</td><td>http://cpdb.molgen.mpg.de/</td><td></td><td></td></tr><tr><td>GO Enrichment Analysis</td><td>http://geneontology.org/page/go-enrichment-analysis</td><td></td><td></td></tr></tbody></table>

sample preparation and analysis of rnaseq-based gene expression data from zebrafish

Die Analyse der Veränderungen der globalen Genexpression ist ein wertvolles Instrument zur Identifizierung von neuartige Wege beobachteten Phänotypen zugrunde liegen. Der Zebrabärbling ist ein hervorragendes Modell für schnelle Beurteilung der ganze Transkriptom vom ganzen Tier oder einzelne Zellpopulationen wegen der Leichtigkeit der Isolierung der RNA aus große Zahl von Tieren. Hier ist ein Protokoll für globale gen Expressionsanalyse in den Zebrafish Embryos mit RNA Sequenzierung (RNASeq) vorgestellt. Wir beschreiben Vorbereitung der RNA aus ganzen Embryonen oder Zell-Populationen mit Zelle Sortierung bei transgenen Tieren erzielt. Wir beschreiben auch einen Ansatz zur Analyse von RNASeq Daten, angereicherte Wege und Gene Ontology (gehen Sie) Begriffe im globalen gen Ausdruck Datensätze zu identifizieren. Zu guter Letzt bieten wir ein Protokoll für die Validierung von Veränderungen der Genexpression mit quantitative Reverse Transkriptase PCR (qRT-PCR). Diese Protokolle können für die vergleichende Analyse der Steuerung und experimentellen Gruppen von Zebrafisch verwendet werden, um Veränderungen der neuartigen Genexpression zu identifizieren und molekulare Einblick in Phänotypen von Interesse.

Sortierung der differenziell ausgedrückt Gene:
Wir gezielte Ermittlung differentiell exprimierten Gene im Larvenstadium von Zebrafisch-Modellen von Alström-Syndrom und Bardet-Bardet-Syndrom (BBS), entweder alms1 oder bbs1 Abschriften durch die Injektion von zuvor validiert Spleiß-blocking MOs in Wildtyp Zebrafish Embryos16,17. 5 Tage nach Befruchtung (D...

Watch this Scientific Journal Video about Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish at JoVE.com

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Probenvorbereitung und Analyse der RNASeq-basierte Genexpressionsdaten von Zebrafisch

Dieses Protokoll stellt einen Ansatz für die ganze Transkriptom-Analyse von Zebrafisch-Embryonen, Larven, oder Zellen sortiert. Wir gehören Isolierung von RNA, Pathway-Analyse von RNASeq Daten und qRT-PCR-basierte Validierung von Veränderungen der Genexpression.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

sample-preparation-analysis-rnaseq-based-gene-expression-data-from

Universidad San Sebastian

Research

JoVE Journal

Genetics

10.8K Aufrufe.  University of Maryland School of Medicine. Dieses Protokoll stellt einen Ansatz für die ganze Transkriptom-Analyse von Zebrafisch-Embryonen, Larven, oder Zellen sortiert. Wir gehören Isolierung von RNA, Pathway-Analyse von RNASeq Daten und qRT-PCR-basierte Validierung von Veränderungen der Genexpression.

Serie: Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Großzebrafisch Embryonale Herz Dissection für Transkriptionsanalyse

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Forschung

Entwicklungsbiologie

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetik

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolierung und Charakterisierung von Einzelzellen aus Zebrafischembryonen

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

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.

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Biologie

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

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.

Globale Genexpressionsanalyse Mit einem Zebrafisch Oligonukleotid-Microarray-Plattform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

<meta charset="utf8"></head><body>Fluoreszierende Proteinmarkierung in Zebrafischlarven mittels CRISPR/Cas9-Knock-in

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Vorbereitung kleiner RNA-Bibliotheken für die Sequenzierung von frühen Mausembryonen

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Aufbereitung von Darmzellen aus Zebrafischlarven für die Einzelzell-RNA-Sequenzierung

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Autor im Rampenlicht: Isolierung und Analyse von Darmzellen von Zebrafischlarven zur Untersuchung transkriptomischer Aspekte der gastrointestinalen Entwicklung 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

Probenvorbereitung und Analyse der RNASeq-basierte Genexpressionsdaten von Zebrafisch

Zusammenfassung

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Großzebrafisch Embryonale Herz Dissection für Transkriptionsanalyse

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

Isolierung und Charakterisierung von Einzelzellen aus Zebrafischembryonen

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

Globale Genexpressionsanalyse Mit einem Zebrafisch Oligonukleotid-Microarray-Plattform

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Vorbereitung kleiner RNA-Bibliotheken für die Sequenzierung von frühen Mausembryonen

Darmisolierung aus Zebrafischlarven für die Einzelzell-RNA-Sequenzierung