Name: targeted rna sequencing assay to characterize gene expression and genomic alterations
Uploaded: 2016-08-04T13:00:00
Description: Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

We give special thanks to Ezra Lyon, Eliot Zhu, Michele Wing, Esko Kautto and Eric Samorodnitsky for technical support. We would also like to thank Jenny Badillo for her administrative support for our team. We acknowledge the Ohio Supercomputer Center (OSC) for providing disk space, processing capacity, and support to run our analyses. We thank the Comprehensive Cancer Center (CCC) at The Ohio State University Wexner Medical Center for their administrative support of this work. S.R. and Team are supported by the American Cancer Society (MRSG-12-194-01-TBG), a Prostate Cancer Foundation Young Investigator Award, NHGRI (UM1HG006508-01A1), Fore Cancer Research Foundation, American Lung Association, and Pelotonia.

Nota: Este protocolo describe el procesamiento y el análisis de cuatro muestras simultánea. Este método es compatible con ARN aislado de células, tejido fresco congelado y tejido incluido en parafina fijado con formalina (FFPE). Este protocolo inicia con 50 - 1000 ng (250 ng recomendado) de iniciar la entrada de ARN para cada muestra. 1. El agotamiento de ARNr y la fragmentación de ARN Procedimiento <ol><li> El agotamiento rRNA <ol><li> Retire eluyen, prima, mezcla de...

<ol>
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Captura RNAseq es una estrategia intermedia entre RNAseq y microarrays enfoques para la evaluación de una parte seleccionada del transcriptoma. Las ventajas de captura incluyen el costo, el tiempo de respuesta rápido redujeron en un secuenciador de escritorio, de alto rendimiento, y la detección de alteraciones genómicas. El método puede ser adaptado para caracterizar los ARN no codificantes 23, detectar solo nucleótido variantes 4-6, examinar empalme de ARN, y para identificar las fusiones d...

Whole transcriptome or RNA sequencing (RNAseq) is an unbiased sequencing method to assess all RNA products. The goal of targeted RNAseq (Capture) is a focused evaluation of selected transcripts with increased sensitivity, dynamic range, reduced cost or scale, and increased throughput compared to standard RNAseq. Similar to standard RNAseq, targeted enrichment approaches can be used to evaluate gene expression, multiple RNA species such as mRNA, microRNA (miRNA), lncRNA1, other noncoding RNAs2, gene fusions3, and mutations4-6.
Capture involves hybridization of complementary oligonucleotides to enrich cDNA libraries for sequencing. The rationale for RNAseq Capture is similar to microarray approaches where complementary oligonucleotides or probes are hybridized to samples and then measured for relative abundance. For microarray technologies, expression is based on relative signal measured for transcripts binding to these probes. Microarrays are thus limited by range, potential background noise from non-specific binding, and cross-hybridization of probes. Furthermore, arrays have limited dynamic range for low and highly expressed transcripts compared to RNAseq1. Microarrays are widely utilized due to their reduced cost and high throughput capacity compared to RNAseq. 
Here, we demonstrate a method for RNAseq Capture that offers a middle ground between RNAseq and microarray approaches for evaluating the transcriptome. RNAseq Capture has intermediate throughput, greater dynamic range and sensitivity, and is scaled for fast turnaround on desktop sequencers. RNAseq Capture also requires reduced computational resources in terms of storage space and data processing.

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			<td height="20" style="height:20px;width:407px;">Thermomixer R</td>
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			<td height="20" style="height:20px;">Centrifuge 5417R</td>
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			<td height="20" style="height:20px;">miRNeasy Mini Kit</td>
			<td style="width:153px;">Qiagen</td>
			<td style="width:143px;">217004</td>
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			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-6X500ML</td>
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			<td height="20" style="height:20px;width:407px;">Thermoblock 24 X 1.5ml</td>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">21516-166</td>
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			<td height="20" style="height:20px;width:407px;">MiSeq Reagent Kit v2 (300-cycles)</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;">MS-102-2002</td>
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			<td height="20" style="height:20px;width:407px;">MiSeq Desktop Sequencer</td>
			<td style="width:153px;">Illumina</td>
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			<td height="20" style="height:20px;width:407px;">PhiX Control v3</td>
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			<td style="width:143px;">FC-110-3001</td>
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			<td height="40" style="height:40px;width:407px;">TruSeq Stranded Total RNA Kit with RiboZero Gold SetA</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;">RS-122-2301</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p5</td>
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			<td>127040822</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(6nt)</td>
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			<td>127040823</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(8nt)</td>
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			<td>127040824</td>
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			<td height="20" style="height:20px;">Agencourt&#174; AMPure&#174; XP - PCR Purification beads&#160;</td>
			<td>Beckman-Coulter</td>
			<td>A63880</td>
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			<td height="20" style="height:20px;">Dynabeads&#174; M-270 Streptavidin</td>
			<td>Life Technologies</td>
			<td>65305</td>
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			<td height="20" style="height:20px;">COT Human DNA, Fluorometric Grade, 1mg</td>
			<td>Roche Applied Science</td>
			<td>05480647001</td>
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			<td height="20" style="height:20px;">Qubit&#174; Assay Tubes&#160;</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
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			<td height="20" style="height:20px;">Qubit&#174; dsDNA HS Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SeqCap&#174; EZ Hybridization and Wash Kits&#160; (24 or 96 reactions)</td>
			<td>Roche NimbleGen&#160;</td>
			<td>05634261001 or 05634253001&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Qubit&#174; 2.0 Fluorometer&#160;</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
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			<td height="20" style="height:20px;width:407px;">10 x 2 ml IDTE pH 8.0 (1X TE Solution)</td>
			<td style="width:153px;">IDT</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tween20 BioXtra</td>
			<td style="width:153px;">Sigma</td>
			<td>P7949-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease Free Water</td>
			<td>Life Technologies</td>
			<td>AM9937</td>
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			<td height="20" style="height:20px;">C1000 Touch&#8482; Thermal Cycler with 96&#8211;Well Fast Rection Module</td>
			<td style="width:153px;">Biorad</td>
			<td style="width:143px;">185-1196</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">SeqCap EZ Hybridization and Wash Kits</td>
			<td>Roche Applied Science</td>
			<td>05634253001</td>
			<td></td>
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			<td height="20" style="height:20px;">SuperScript II Reverse Transcription 200U/ul</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
			<td></td>
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			<td height="20" style="height:20px;">D1000 ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5582</td>
			<td></td>
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			<td height="20" style="height:20px;">Agencourt RNAClean XP -40ml</td>
			<td>Beckman Coulter Inc</td>
			<td>A63987</td>
			<td></td>
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			<td height="20" style="height:20px;">RNA ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5576</td>
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			<td height="20" style="height:20px;">RNA ScreenTape Ladder</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5578</td>
			<td></td>
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			<td height="20" style="height:20px;">RNA ScreenTape Sample Buffer</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5577</td>
			<td></td>
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			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>Sigma</td>
			<td align="left">72068-100ML</td>
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			<td>Life Technologies</td>
			<td>65602</td>
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			<td height="20" style="height:20px;">DYNAMAG -96 SIDE EACH</td>
			<td>Life Technologies</td>
			<td>12331D</td>
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			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432-1L</td>
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			<td height="20" style="height:20px;">KAPA HotStart ReadyMix</td>
			<td>KAPA Biosystems</td>
			<td>KK2602</td>
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			<td height="20" style="height:20px;width:407px;">NanoDrop 2000 Spectrophotometer</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:407px;">My Block Mini Dry Bath</td>
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			<td style="width:153px;">Agilent Technol. Inc.</td>
			<td>5067- 5583</td>
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			<td height="20" style="height:20px;width:407px;">Vacufuge Plus</td>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">022829861&#160;</td>
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targeted rna sequencing assay to characterize gene expression and genomic alterations

la secuenciación de ARN (RNAseq) es un método versátil que puede ser utilizado para detectar y caracterizar la expresión génica, las mutaciones, las fusiones de genes, y noncoding RNAs. Estándar RNAseq requiere 30 - 100 millones de secuenciación lee y puede incluir varios productos de ARN como ARNm y ARN no codificantes. Demostramos cómo orientar las RNAseq (captura) permite un estudio centrado en productos de ARN seleccionados utilizando un secuenciador de escritorio. captura RNAseq puede caracterizar transcripciones anotadas, bajas, o expresado de forma transitoria que de otra manera no pueden ser detectados usando métodos tradicionales RNAseq. Aquí se describe la extracción de ARN a partir de líneas celulares, el agotamiento de ARN ribosomal, síntesis de ADNc, la preparación de bibliotecas de código de barras, la hibridación y la captura de las transcripciones específicas y la secuencia multiplex en un secuenciador de escritorio. También describimos la tubería análisis computacional, lo que incluye la evaluación del control de calidad, la alineación, la detección de la fusión, la cuantificación de la expresión génica y la identificación de un solo nucleotide variantes. Este ensayo permite la secuenciación de transcripción específico para caracterizar la expresión génica, fusiones de genes, y las mutaciones.

A destacando esquemática pasos clave en Capture RNAseq se muestra en la Figura 1. Cuatro líneas celulares de cáncer con mutaciones conocidas se utilizaron para demostrar la eficacia de la técnica de RNAseq Capture (K562 con la fusión ABL1, LC2 con la fusión RET, EOL1 con la fusión PDGFRalpha y RT- 4 con la fusión FGFR3). Las cuatro muestras se agruparon y se secuenciaron con 2x 100 pb lee en un secuenciador de escritorio, que g...

Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

La secuenciación de ARN dirigidos Ensayo para caracterizar la expresión génica y la genómica alteraciones

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Investigación

Biología

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

Aislamiento del ARN de síntesis embrionarias pez cebra y el cDNA de análisis de expresión génica

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Competitive Genomic Screens of Barcoded Yeast Libraries

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

La detección de alteraciones genéticas somáticas en muestras de tumores por Exon de captura y secuenciación masivamente paralelo

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...