Name: rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord
Uploaded: 2014-11-01T14:00:00
Description: Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1. electroporate konstruktionen av intresse i neurala Rör av HH12-13 Chicken embryon i ovo Använd en fluorescerande reporter, helst i ett bicistronisk avskrift eller smält till proteinet av intresse med en intercalating viral 2a peptid 11, för att möjliggöra snabb identifiering av individer med tillfredsställande nivåer av transfektion. OBS: Typisk inkubationstid för denna etapp är ca 48 timmar vid 37,8 ° C. <ol><li> Öppna ett litet fönster i äggen efter a...

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1 (8), 207-20 (1999).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Dev. Dyn. 229 (3), 433-439 (2004).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> J. Vis. Exp. (9), 10-3791 (2007).</li><li>Sauka-Spengler, T., Barembaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+approaches+in+the+chick+embryo.">Gain- and loss-of-function approaches in the chick embryo.</a> Methods Cell Biol. 87, 237-256 (2008).</li><li>Ghadge, G. D., 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=Truncated+wild-type+SOD1+and+FALS-linked+mutant+SOD1+cause+neural+cell+death+in+the+chick+embryo+spinal+cord.">Truncated wild-type SOD1 and FALS-linked mutant SOD1 cause neural cell death in the chick embryo spinal cord.</a> Neurobiol. Dis. 21 (1), 205-20 (2006).</li><li>Megason, S. G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mitogen+gradient+of+dorsal+midline+Wnts+organizes+growth+in+the+CNS.">A mitogen gradient of dorsal midline Wnts organizes growth in the CNS.</a> Development. 129 (9), 2087-2098 .</li><li>Swartz, M., Eberhart, J., Mastick, G. S., Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparking+new+frontiers:+using+in+vivo+electroporation+for+genetic+manipulations.">Sparking new frontiers: using in vivo electroporation for genetic manipulations.</a> Dev. Biol. 233 (1), 13-21 (2001).</li><li>Cloonan, N., 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=Stem+cell+transcriptome+profiling+via+massive-scale+mRNA+sequencing.">Stem cell transcriptome profiling via massive-scale mRNA sequencing.</a> Nat. Methods. 5 (7), 613-619 (2008).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nat. Methods. 5 (7), 621-628 (2008).</li><li>Goecks, J., Nekrutenko, A., Taylor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy+Team.+Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), 86 (2010).</li><li>Trichas, G., Begbie, J., Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+viral+2A+peptide+for+bicistronic+expression+in+transgenic+mice.">Use of the viral 2A peptide for bicistronic expression in transgenic mice.</a> BMC Biol. 6, 40 (2008).</li><li>Maniatis, T., Fritsch, E. F., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning. A laboratory manual. , (1982).</li><li>Wang, 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=Evaluation+of+the+coverage+and+depth+of+transcriptome+by+RNA-Seq+in+chickens.">Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens.</a> BMC Bioinformatics. 12, 10-1186 (2011).</li><li>Blankenberg, D., 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=Manipulation+of+FASTQ+data+with+Galaxy.">Manipulation of FASTQ data with Galaxy.</a> Bioinformatics. 26 (14), 1783-1785 (2010).</li><li>Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biol. 14 (4), 36 (2013).</li><li>Trapnell, C., 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=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat Biotechnol. 28 (5), 511-515 (2010).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Vieceli, F. M., Sim&#245;es-Costa, M., Turri, J. A., Kanno, T., Bronner, M., Yan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+chicken+Scratch2+is+expressed+in+a+subset+of+early+postmitotic+neural+progenitors.">The transcription factor chicken Scratch2 is expressed in a subset of early postmitotic neural progenitors.</a> Gene Expr. Patterns. 13 (5-6), 189-196 (2013).</li><li>Chesnutt, C., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid-based+short-hairpin+RNA+interference+in+the+chicken+embryo.">Plasmid-based short-hairpin RNA interference in the chicken embryo.</a> Genesis. 39 (2), 73-78 (2004).</li><li>Katahira, T., Nakamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+in+chick+embryos+with+a+vector-based+small+interfering+RNA+system.">Gene silencing in chick embryos with a vector-based small interfering RNA system.</a> Dev. Growth. Differ. 45 (4), 361-367 (2003).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231 (3), 592-600 (2004).</li><li>Lu, Y., Lin, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transgenic+strategies+in+the+developing+chicken+spinal+cord.">PiggyBac transgenic strategies in the developing chicken spinal cord.</a> Nucleic Acids Res. 37 (21), 141 (2009).</li><li>Sim&#245;es-Costa, M., Tan-Cabugao, J., Antoshechkin, I., Sauka-Spengler, T., Bronner, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+reveals+novel+players+in+the+cranial+neural+crest+gene+regulatory+network.">Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network.</a> Genome Res. 24 (2), 281-290 (2014).</li><li>Trapnell, C., 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=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat. Protoc. 7 (3), 562-578 (2012).</li></ol>

Här ger vi riktlinjer för att analysera effekter efter elektroporering av hönan ryggmärgen. Även elektroporering av DNA-vektorer är oftare används för att överuttrycker en gen av intresse, kan man även använda konstruktioner som kodar dominerande negativ, quimeric proteiner eller prekursorer för siRNA att generera geners funktion knock-down förhållanden 19-21. I själva verket kan jämföra transkriptom profiler som följer av både gain och förlust av funktionsanalys påpeka gener differentiel...

Genetiska studier av levande organismer använder ofta kyckling embryot som modell eftersom i ovo elektroporering representerar ett snabbt och billigt sätt att delvis transfektera embryonala strukturer in vivo med DNA-konstruktioner 1-5. Bicistronisk expressionsvektorer som kodar för ett transkript som innehåller en fluorescerande reporter tillsammans med genen av intresse, såsom pCIG 6 eller pMES 7, möjliggöra snabb kontroll av transfektion kvalitet i den önskade regionen under ett stereomikroskop innan bearbetning embryon för nedströms analys. Med senaste framstegen inom DNA-sekvensering, är det nu möjligt att få digitala hela transkriptom uttryck profiler från RNA-prover som använder RNA-Seq 8,9. I stället för tidskrävande metoder som möjliggör analys av endast ett fåtal genprodukter parallellt, till exempel in situ hybridisering, immunohistokemi och qPCR, utarbetande av cDNA-bibliotek förhög genomströmning sekvensering kan ge information om hela transkriptom. Observation av de experimentella effekter på uttrycksnivåer av varje enskild gen kan i sin tur ge kraftfulla insikter om banorna förändras av konstruktionen används. Detta är särskilt lätt om en genomisk aggregat för den använda organismen är tillgänglig, återigen är sålunda kycklingembryo en lämplig modell. Om användaren har tillgång till en tillförlitlig genomsekvense centrum är den viktigaste frågan att få hög kvalitet RNA-prover. Detta arbete visar en metod för att erhålla RNA-prov från transfekterade neurala rören lämpliga för RNA-Seq, liksom de efterföljande stegen för in silico-analys av de resulterande datamängderna. Efter elektroporering vid HH12-13 följt av 48 timmars inkubation, transfekterades trunkryggmärgs halvor dissekerades i ribonukleas fria betingelser, omedelbart lyserades och ytterligare skyddas från nedbrytning genom ultra-låg frys tills RNA-rening och bibliotek Förberedelsejon. Galaxy allmän server 10 ger tillgång till fria resurser för att analysera hög genomströmning sekvenseringsdata. Det utrymme som erbjuds för registrerade användare är nog för små experiment, vilket eliminerar behovet av lokal beräknings infrastruktur. RNA-Seq dataanalys inkluderar filtrering, anpassning och kvantifiering / jämförelse av genuttryck. Även om det finns allmänna riktlinjer för RNA-Seq dataanalys, kommer filtreringsparametrar beror till stor del på kvaliteten på sekvensering och bör bestämmas efter kvalitetskontroll analys av rådata. Detta protokoll beskriver stegen för att få och jämföra RNA-Seq profiler från kyckling embryonal ryggmärg transfekterade in vivo. Som ett representativt resultat, jämförelse av datauppsättningar som erhållits från två oberoende kontrollprover transfekterade med en tom vektor visade att metoden är reproducerbar och bör göra det möjligt att identifiera differentiellt ExpresSED-transkript i experimentprover. Därför har tillämpningen av denna metod potentialen att kraftigt öka antalet upptäckter efter ett enda experiment och ger därmed en stor uppsättning data för efterföljande undersökning.

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Indian &#236;nk</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18G x 1 1/2 needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 ml syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>MP Biomedicals</td>
			<td>819620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F D &#38; C Blue 1</td>
			<td>Spectra Colors Corporation</td>
			<td>5.FC.0010P0</td>
			<td>Food Dye used to visualize DNA injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ringer&#39;s solution (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 0.37 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.225 g CaCl2.2H2O</td>
			<td>Fisher Scientific</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.217 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.02 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Filter and autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (Phosphate Buffered Solution) (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 8 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.2 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 1 ml DEPC (Diethylpyrocarbonate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Shake vigorously and let stand overnight at room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 60 x 15 mm polystirene Petri dishes</td>
			<td>Corning Life Sciences</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight fine point tweezers</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td>
			<td>Kimble Chase</td>
			<td>40A502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manual pipette pump</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clear 1.5 ml microcentrifuge polypropilene tubes</td>
			<td>Corning Life Sciences</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAlater solution for RNA stabilization</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAqueous total RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1912</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Truseq RNA Sample Preparation Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001/RS-122-2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

För att validera den metod som beskrivs, har två kontrollprover som genereras från embryon elektroporerade med den tomma vektorn pMES 7 och ett prov från embryon som transfekterats med samma vektor innehållande insatsen konstruera SCRT2-ZNF, som kodar de zink-fingerdomäner av kyckling SCRT2 18. Prover som ger 11-22 mikrogram totalt RNA vardera erhölls från pooler av 8 till 12 embryon. Samtliga tre prover gjorde ett optimalt RIN värde av 10 i en kvalitetskontroll utförs med Bioanalyzer ins...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq analys av Differential genuttryck i elektro Chick embryonala Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Dissektion och kultur commissural neuroner från embryonala ryggmärg

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Ryggmärgen Elektrofysiologi II: Extracellulär Sug elektrod Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Dissekering och kultur Chick Statoacoustic Ganglion och Spinal explants sladd i Collagen Gel för Neurite utväxt analyser

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Dii-märkning av DRG nervceller att Studera axonal Förgrening i en hel Mount Beredning av Mouse Embryonala ryggmärg

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

In ovo Expression of MicroRNA in Ventral Chick Midbrain

I ovo Uttryck av MikroRNA i ventrala Chick mitthjärnan

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Stereotaktisk injektion av en viral vektor för villkorad Gene Manipulation i ryggmärgen hos möss

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

Dissection och Lateral Montering av Zebrafish embryon: Analys av ryggmärgs Utveckling

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Spinal Cord Transection in the Larval Zebrafish

Spinal Cord transection i Larvernas Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

En<em&gt; Ex vivo</em&gt; Laser-inducerad ryggmärgsskada modell för bedömning Mekanismer axonal degeneration i realtid

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Adeno-associerade Virus-medierad transgenens uttryck i genetiskt definierade nervceller i ryggmärgen

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...