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

This work was supported by R01DK102001 (N.A.Z.), P30DK072488 (N.A.Z.), and T32DK098107 (T.L.H. and J.E.N.).

All animal protocols outlined below are in accordance with and approved by the University of Maryland Institutional Animal Care and Use Committee (IACUC).
1. Embryo Preparation
<ol>
	<li>Generating embryos through natural mating
	<ol>
		<li>Culture embryos to 3 months of age, reproductive maturity5,8.</li>
		<li>Segregate adult male and female fish from the desired strain into divided mating tanks on the evening befo...

<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>

The approach described in this protocol offers a relatively rapid and cost-effective strategy for transcriptome-level analysis of whole animals or specific sorted cell populations. The zebrafish provides an advantageous model for this type of study because of the ease and rapidity in generating large amounts of starting material, the ease of implementing genetic or environmental experimental conditions, and the availability of a large spectrum of transgenic reporter lines allowing for isolation of cell-type specific and ...

Comparative analysis of global gene expression is a valuable tool to identify novel genes contributing to observed phenotypes. Such analyses typically rely on quantitative assessment of transcript abundance compared between experimental and control samples. Targeted approaches, such as qRT-PCR are relatively rapid and accurate for investigation of single gene expression changes. RNA sequencing (RNASeq) offers a broad, hypothesis-free approach to identify significant changes in gene expression between samples, making it now the standard for such investigations across experimental systems.
Zebrafish have emerged as a prominent model across many disease areas. Originally developed for their utility in developmental biology studies, due to their high fecundity and relatively low cost of maintenance, experimental use of zebrafish has evolved to include a broad range of phenotypes from embryonic to adult stages as well as a wide array of molecular assays1,2,3. Indeed, these advantages make molecular mechanistic studies rapid and cost-effective because of the ease of acquiring large amounts of material combined with the ease of both genetic and environmental manipulation at all stages of life. Moreover, the transparent nature of zebrafish embryos and larvae make it ideal for generating cell- and tissue-specific transgenic reporter lines allowing in vivo visualization of discrete cell populations4. Exploitation of such lines permits global gene expression analysis in specific isolated cell types based on reporter gene expression.
Here we present a comprehensive protocol for global gene expression analysis using RNASeq after culture of zebrafish embryos. Genetic experimental manipulations, including morpholino (MO)-based transient gene knockdown or CRISPR-mediated genome editing, have been presented elsewhere5,6,7. We therefore focus on a detailed protocol for isolation of RNA from whole embryos or sorted transgenic reporter-expressing cells followed by simple computational analysis of RNASeq results using pathway tools and gene ontology (GO) terms. Finally, we have included a strategy for validation of gene expression changes by quantitative reverse transcriptase PCR (qRT-PCR). These protocols are applicable to zebrafish embryos subjected to a wide range of experimental conditions, including comparison of genetic mutants or environmental conditions.

<table>
	<tbody>
		<tr>
			<td>Commercial Reagents</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Zebrafish Strains</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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 colspan="2">http://geneontology.org/page/go-enrichment-analysis</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

Sorting of Differentially Expressed Genes:
To identify differentially expressed genes in the larval stage of zebrafish models of Alstr&#246;m Syndrome and Bardet-Biedl Syndrome (BBS), we targeted either alms1 or bbs1 transcripts by injecting previously validated splice-blocking MOs into wild-type zebrafish embryos16,17. At 5 days post fertilization (dpf), ...

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

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

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

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