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 den Construct av interesse i Neural Rør av HH12-13 Chicken embryoene i ovo Bruk en fluorescerende reporter, fortrinnsvis i en bicistronisk avskrift eller smeltet til protein av interesse med en interkalerende viral 2a peptid 11, for å muliggjøre rask identifisering av personer med tilfredsstillende nivåer av transfeksjon. MERK: Typisk Inkubasjonstiden for dette trinn er rundt 48 timer ved 37,8 ° C. <ol><li> Åpne et lite vindu i eggene etter fjerni...

<ol>
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Her gir vi retningslinjer for å analysere effekter etter elektroporering av kylling ryggmargen. Selv elektroporering av DNA vektorer blir oftere brukt til å overuttrykke et gen av interesse, kan man også bruke konstruksjoner som koder dominerende negativer, quimeric proteiner eller forløpere for sirnas å generere genfunksjon knock-down forhold 19-21. Faktisk kan transkriptom sammenligning av profiler som følge av både gevinst-og tap-av-funksjon-analyse påpeke gener som uttrykkes differensielt i motsat...

Genetiske studier på levende organismer ofte bruker kylling embryo som modell fordi i ovo elektroporering representerer en rask og rimelig måte å delvis transfektere embryonale strukturer in vivo med DNA konstruerer 1-5. Bicistronisk ekspresjonsvektorer som koder for en transkript inneholdende et fluoriserende reporter sammen med genet av interesse, slik som pCIG pMES 6 eller 7, tillater rask verifisering av transfeksjon kvalitet i det ønskede område under et stereomikroskop før behandlingen embryoer for genetisk analyse. Med de siste fremskritt innen DNA-sekvensering, er det nå mulig å få digitale hele transkriptom uttrykk profiler fra RNA prøver ved hjelp av RNA-Seq 8,9. Derfor, i stedet for tidkrevende fremgangsmåter som muliggjør analyse av bare noen få genprodukter i parallell, så som in situ hybridisering, immunhistokjemi og qPCR, fremstilling av cDNA-biblioteker forhigh-throughput sekvensering kan gi informasjon av hele transkriptomet. Observasjon av de eksperimentelle effekter på ekspresjonsnivåer av hver enkelt gen kan i sin tur gi kraftige innsikt om trasé endret ved konstruksjonen anvendes. Dette er spesielt lett hvis en genomisk sammenstilling for den organisme som anvendes er tilgjengelig, igjen er således chick embryo en passende modell. Hvis brukeren har tilgang til en pålitelig genomsekvense sentrum den viktigste saken å oppnå høy kvalitet RNA prøver. Dette arbeidet viser en fremgangsmåte for oppnåelse av RNA-prøver fra transfekterte nevrale rør som er egnet for RNA-Seq, samt nedstrøms for fremgangsmåten i silico-analyse av de resulterende datasett. Etter elektroporering på HH12-13 etterfulgt av 48 timers inkubasjon ble transfektert trunk ryggmargs halvdeler dissekert i ribonuklease frie forhold, umiddelbart lysert og videre beskyttet mot nedbrytning av ultra-lavt fryse før RNA rensing og bibliotek forberedelse;ion. The Galaxy offentlig server 10 gir tilgang til gratis ressurser for å analysere high-throughput sekvense data. Mengden plass tilbudt for registrerte brukere er nok for små eksperimenter, og dermed eliminere behovet for lokal beregnings infrastruktur. RNA-Seq dataanalyse inkluderer filtrering, justering og kvantifisering / sammenligning av genuttrykk. Selv om det er generelle retningslinjer for RNA-Seq dataanalyse, vil filtreringsparametre i stor grad avhenge av kvaliteten på sekvensering og bør bestemmes etter kvalitetskontroll analyse av rådata. Denne protokollen beskriver fremgangsmåten for å innhente og sammenligne RNA-Seq profiler fra kylling embryonale ryggmargen transfektert in vivo. Som et representativt resultat av sammenligning av datasett oppnådd fra to uavhengige kontrollprøver transfektert med tom vektor viste at metoden er reproduserbar, og bør tillate identifisering av differensielt expresaliserte utskrifter i eksperimentelle prøver. Derfor har anvendelsen av denne fremgangsmåte potensial til å betydelig øke antall oppdagelser etter en enkelt eksperiment, og således tilveiebringe et stort sett av data for etterfølgende undersøkelse.

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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
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		<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>
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		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
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		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
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			<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>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Shake vigorously and let stand overnight at room temperature</td>
			<td></td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
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			<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>
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			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Straight fine point tweezers</td>
			<td>Any supplier</td>
			<td></td>
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			<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>
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			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
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			<td height="20" style="height:20px;">Manual pipette pump</td>
			<td>Any supplier</td>
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			<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>
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			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
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		<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>
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			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
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		<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.

For å validere metoden beskrevet, ble to kontrollprøver genereres fra embryoer elektroporerte med de tomme vektor pMES 7 og en prøve fra embryoer transfektert med samme vektor som inneholder innsatsen konstruere SCRT2-ZNF, som koder for de sink-finger domener av kylling SCRT2 18. Prøver som ga 11-22 mikrogram total RNA hvert ble hentet fra bassenger av 8 til 12 embryoer. Alle tre prøvene scoret RIN en optimal verdi på 10 i en kvalitetssjekk utført med Bioanalyzer instrumentet (figur ...

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 Analyse av Differential Gene Expression i elektroporerte 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 ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

Disseksjon og kultur av Chick Statoacoustic ganglion og Spinal Cord explants i Collagen Gels for neurite utvekst Assays

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-Merking av DRG Neurons til Study aksonal Avgrening i en hel Mount Utarbeidelse av Mouse Embryonic Spinal Cord

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 Expression av mikroRNA i ventral Chick midthjernen

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

Stereotaksiske Injeksjon av en viral vektor for betinget genmanipulering i Mouse Spinal Cord

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

Disseksjon og Lateral Montering av sebrafisk embryo: Analyse av ryggmargs Development

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 tran i Larve Sebrafisk

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-indusert ryggmargsskade Model å vurdere Mekanismer av aksonal degenerasjon i sanntid

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-assosiert Virus-mediert Transgene uttrykk i genetisk definerte nerveceller i ryggmargen

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