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 HH12-13 닭 배아의 신경 튜브 OVO의 관심의 구조물 형질 전환의 만족 수준이 개인의 빠른 식별을 가능하게하기 위해, 바람직하게는 시스 트론 성적 증명서, 형광 기자를 사용하거나 인터 바이러스 2A 펩타이드 (11)와 함께 관심의 단백질에 융합. 참고 :이 단계에 대한 일반적인 배양 시간은 37.8 ° C에서 약 48 시간이다. <ol><li> 18 G X 1 1/2 바늘에 연결된 ?...

<ol>
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여기에서 우리는 닭 척수의 전기 후 효과 분석을위한 지침을 제공합니다. DNA 벡터의 전기가 더 자주 관심의 유전자를 과발현하는 데 사용되지만, 하나는 또한 유전자 기능 녹다운 조건 19-21를 생성하는 siRNA를 위해 우성 네거티브 quimeric 또는 전구체 단백질을 코딩하는 구조를 이용할 수있다. 실제로, 이득 - 및 기능 상실 분석 모두에 기인 사체 프로파일의 비교는 차별적 따라서 전위가 더 ...

OVO 전기에 DNA 1-5을 구성하여 부분적으로 생체 내에서 배아 구조에 감염 될 수있는 빠르고 저렴한 방법을 나타 내기 때문에 살아있는 유기체에 유전 연구는 자주 모델로 닭 배아를 사용합니다. 이러한 pCIG 6 pMES 7과 같은 목적 유전자와 함께 형광 리포터를 함유 한 증명서를 인코딩 시스 트론 발현 벡터, 하류 분석 배아를 처리하기 전에 실체 현미경을 이용하여 원하는 영역에 형질 전환 품질의 빠른 확인을 허용한다. DNA 시퀀싱 최근 발전으로, 그것은 RNA-SEQ 8,9을 사용하여 RNA 샘플들로부터 디지털 전 사체 발현 프로파일을 획득하는 것이 가능하다. 따라서, 대신 그러한 계내 혼성화, 면역 조직 화학 및 qPCR에,의 cDNA 라이브러리의 제조에서와 같이 병렬로 몇 유전자 산물의 분석을 가능하게 시간이 걸리는 방법높은 처리량 시퀀싱 전 사체의 정보를 제공 할 수있다. 모든 단일 유전자의 발현 수준에 대한 효과 실험 관찰 차례로 사용 구조체에 의해 변경 경로에 강력한 통찰력을 제공 할 수있다. 이 사용 유기체의 게놈 어셈블리를 사용할 수있는 경우 특히 쉽고, 따라서 병아리 배아는 다시 적당한 모델이다. 사용자가 신뢰할 수있는 게놈 시퀀싱 센터에 액세스하는 경우, 주요 문제는 고품질의 RNA 샘플을 획득한다. 본 연구는 생성 된 데이터 세트의 실리코 분석을 위해 RNA-SEQ 적합한 형질 신경 튜브뿐만 아니라 하류 단계로부터 RNA 샘플을 얻는 방법을 설명한다. 48 시간 배양 한 다음 HH12-13에 전기 한 후, 형질 전환 된 트렁크 척수 절반은, 리보 뉴 클레아가없는 상태에서 해부 즉시 용해 및 추가 RNA 정화 및 라이브러리는 준비 될 때까지 매우 낮은 동결에 의해 분해로부터 보호했다이온. 갤럭시 공공 서버 (10)는 높은 처리량 시퀀싱 데이터 분석을위한 무료 자원에 대한 액세스를 제공합니다. 등록 된 사용자를 위해 제공되는 공간의 크기는 국부적 계산 된 기반 구조에 대한 필요성을 제거, 소형 실험에 충분하다. RNA-SEQ 데이터 분석 필터링, 정렬 및 유전자 발현의 정량 / 비교를 포함한다. RNA-SEQ 데이터 분석에 대한 일반적인 가이드 라인이 있지만, 필터링 파라미터 시퀀싱의 품질에 크게 의존 할 것으로 예상되며, 원 데이터의 품질 관리 분석을 통해 결정되어야한다. 이 프로토콜은 획득 및 생체 내에서 형질 전환 닭의 배아 척수에서 RNA-SEQ 프로파일을 비교하는 단계에 대해 설명합니다. 대표적인 결과, 빈 벡터로 형질 개의 독립 제어 샘플들로부터 얻어진 데이터 세트의 비교 방법은 재현성 및 차등 EXPRES의 식별을 허용한다는 것을 보여 주었다실험 샘플에 나오지도 성적 증명서. 따라서, 이러한 방법의 응용이 크게 하나의 실험 후에 발견의 수를 증가하고, 따라서 후속 조사를 위해 큰 데이터 세트를 제공하는 잠재력을 가지고있다.

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			<td>Any supplier</td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
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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>
			<td></td>
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		<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>
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			<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>
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			<td></td>
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		<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>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
			<td></td>
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		</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>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
			<td></td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight fine point tweezers</td>
			<td>Any supplier</td>
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		</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>
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		<tr height="20">
			<td height="20" style="height:20px;">RNAqueous total RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1912</td>
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			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Any supplier</td>
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			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
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			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
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		</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.

설명 된 방법을 검증하기 위해, 두 개의 제어 샘플은 빈 벡터 pMES 7 삽입 닭 SCRT2 (18)의 아연 손가락 도메인을 인코딩 SCRT2-ZNF를, 구성이 포함 된 동일한 벡터로 형질 전환 된 배아에서 하나의 샘플에 electroporation하여 배아에서 발생했다. 각 11-22 μg의 총 RNA를 산출 샘플은 8 ~ 12 배아의 풀에서 얻었다. 모든 3 개의 시료 고품질 RNA이 프로토콜로 얻을 수있는 것을 보여주는 Bioanalyzer 기기 ...

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.

electroporation하여 병아리 배아 척수에서 차동 유전자 발현의 RNA-SEQ 분석

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

배아 척수에서 뉴런의 Commissural 해부와 문화

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

연구

신경과학

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

척수 전기 생리학 II : 세포 흡입 전극 제작

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

Neurite 가지의 Assays에 대한 해부와 칙 Statoacoustic 신경절과 콜라겐 젤에서 척수의 Explants의 문화

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

마우스 배아 척수의 전체 마운트 준비 분기 Axonal 공부 DRG 뉴런의 DiI - 라벨링

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

복부 병아리 중뇌에있는 마이크로 RNA의 오보, 식

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

마우스 척추 조건부 유전자 조작을위한 바이러스 성 벡터의 Stereotaxic 주입

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

해부 및 제브라 피쉬 배아의 측면 장착 : 척수 개발의 분석

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

애벌레 Zebrafish의 척수 절개

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

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

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

척수의 유전으로 정의 된 신경에 있는 Adeno 관련 바이러스 중재 Transgene 식

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