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.

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S.R. receives funding from Novartis and Ariad Pharmaceuticals for conduct of clinical trials. S.R. immediate family members own stock in Johnson and Johnson.

RNA-Seq Capture ist eine Zwischen Strategie zwischen RNA-Seq und Microarray-Ansätze für einen ausgewählten Teil der Transkriptom-Auswertung. Die Vorteile von Capture beinhalten Kosten, schnelle Durchlaufzeit auf einem Desktop-Sequenzer, einen hohen Durchsatz und der Nachweis genomischer Veränderungen reduziert. Das Verfahren angepasst werden kann , nicht-kodierenden RNAs 23, erkennen single nucleotide zu charakterisieren Varianten 4-6, RNA - Splicing untersuchen und 24 Genfusionen oder strukturelle Umlagerungen zu identifizieren. Weiterhin kann dieser Ansatz zur klinischen oder verarbeiteten Proben angewendet werden , die 24,25 Fixierung mit Formalin und eingebettet in Paraffin Blöcken unterzogen wurden. Es gibt mehrere bedeutende Vorteile von RNA-Seq capture im Vergleich zu Mikroarray, Real-time quantitative PCR, Sanger-Sequenzierung und DNA-Sequenzierung. Microarray zeichnet sich durch hohe Hintergrund begrenzt durch Kreuzhybridisierung und nicht-spezifischen Sonden binden. Quantifizierung von Genen, die mit low Ausdruck ist wegen Hintergrundrauschen beschränkt, während stark exprimierte Gen Messungen von Signalsättigung 1 betroffen sind. Im Vergleich zu RNA-Seq-Capture erweist sich real-time PCR schwierig zu reproduzieren. Zusätzlich RNA-Seq zur Erkennung von neuer Transkripte ermöglicht, erfordert weniger Ausgangsinputmaterial und kann alternatives Spleißen 26 .Im Gegensatz zu Sanger - Sequenzierung erfassen, ermöglicht RNA-Seq für einen höheren Durchsatz und Analyse von niedrigen ausgedrückt miRNA. Sanger-Sequenzierung hat sich als ein wertvolles Werkzeug für die Überprüfung der Fusionen mit bekannten Exon-Exon-Verbindungen und somatische Mutationen DNA sein, aber Identifizierung neuer Fusionen von Anforderungen eines priori Kandidatenunterbrechungs behindert wird. DNA-Sequenzierung ist nicht kosteneffizient, erfordert größere Speicherkapazität für Daten, und unfähig ist, Nachweis von post-transkriptionalen Modifikationen. Es gibt mehrere wichtige Schritte in RNA-Seq-Capture beteiligt. Erstens, zur Verbesserung der Ausbeute von Library-Produkte aus derRNA / cDNA-spezifischen paramagnetischen Perlen und paramagnetischen Kügelchen während Waschungen werden nicht vorsichtig die Perlen über trocken, was zu Ausbeuteverlust führen wird. Auch nicht unter trocknen die Kügelchen, sicherzustellen, dass alle von Ethanol aus den Probenröhrchen, wie Ethanol entfernt wird, kann cDNA Ausbeute verringern. Zweitens ist die Hybridisierung von cDNA - Bibliotheken mit komplementären Sonden abhängig von gleich bleibende Temperatur, wir empfehlen Erwärmung Waschpuffer I und Strikte Buffer auf 65 ° C für mindestens zwei Stunden im Voraus. Ferner wird nach der Hybridisierung ist es wesentlich , während des Bindens und Waschschritte 65 ° C zu halten. Die Sonden hier verwendet wurden für die Exons von Genen von Interesse für die Entwicklung von Arzneimitteln entwickelt, einschließlich Kinasen, Gene gemeinsam Umlagerungen wie Transkriptionsfaktoren beteiligt sind, und Housekeeping-Genen. Außerdem Gen Inhalt ist anpassbar und Capture-Panel-Größen variieren kann. als neue Informationen über Genomregionen Weiterhin entsteht, können zusätzliche Sonden entwickelt und hinzugefügt werden, um ter bestehende Aufnahmetafel. Auswertung der Ausrichtung Metriken, die speziell auf Zielkurs, liefert Informationen darüber, wie gut die Zielregion angereichert wurde. Der Low-Zielrate auf eine fehlgeschlagene Hybridisierung und Einfangen zurückgeführt werden kann, wodurch die gewünschte Zielregion nicht erfasst und angereichert. In diesem Fall wird eine erneute Hybridisierung und Erfassung der Bibliothek Satz muss durchgeführt werden. Der Low-Zielrate kann auch durch Ausfall sein rRNA zu verarmen, die durch Berechnung des Prozentsatzes der rRNA in der Probe bestätigt werden kann. Hohe rRNA Prozentsatz innerhalb der Probe wird erneut Vorbereitung der Probe erfordern mit rRNA Depletion beginnen. Außerdem, wenn Bibliotheks Konzentration unter den Anforderungen für die Hybridisierung und Einfangen fällt, wäre es ratsam, die Menge des Ausgangs Eingang für den Probentyp und die Qualität (Bereich 50-1000 ng) zu optimieren. Zwar gibt es mehrere Vorteile zur gezielten RNA-Seq-Anwendungen sind, gibt es auch EinschränkungenErwägen. Proben mit schlechter Qualität RNA basierend auf RIN oder der Grad der Fragmentierung kann nicht die Qualität Bibliotheken für die Sequenzierung ergeben. Mehrere Gruppen Erfolg mit Formalin fixierten und in Paraffin eingebetteten Proben unter Beweis gestellt haben, aber es gibt Beispiele , die nicht 24,25,27 für die Sequenzierung passieren. da RNA-Seq Capture auf bekannte Transkripte Weiterhin konzentriert, verliert sie die Vorteile der unvoreingenommene RNA-Seq für neue oder nicht-Transkripte. Darüber hinaus ist für SNP-Nachweis können RNA-Seq Methoden nur Mutationen in exprimierten Transkripte nachzuweisen. Zukunftschancen von RNA-Seq-Capture umfassen Forschung und klinische Anwendungen. Kürzliche Entdeckung von Tausenden von langen nicht-kodierenden RNA und ihre Rolle in der Biologie wird fokussierter Charakterisierung erforderlich. In der Klinik kann RNA-Seq-Capture über Forschungstests erweitern und übersetzen in die klinische Assays menschlicher Krankheiten wie Krebs, Infektionskrankheiten, und nicht-invasive Tests zu charakterisieren. In Verbindung mit der genomischen Sequenzierung approaChes können RNA-Seq-Capture integriert werden, um das zum Ausdruck Genom zu untersuchen und zu charakterisieren.

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.

<table border="0" cellpadding="0" cellspacing="0" width="870">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:407px;">Thermomixer R</td>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">21516-166</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5417R</td>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">5417R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">miRNeasy Mini Kit</td>
			<td style="width:153px;">Qiagen</td>
			<td style="width:143px;">217004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-6X500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:407px;">MiSeq Desktop Sequencer</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:407px;">PhiX Control v3</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;">FC-110-3001</td>
			<td></td>
		</tr>
		<tr height="40">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p5</td>
			<td style="width:153px;">IDT</td>
			<td>127040822</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(6nt)</td>
			<td style="width:153px;">IDT</td>
			<td>127040823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(8nt)</td>
			<td style="width:153px;">IDT</td>
			<td>127040824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt&#174; AMPure&#174; XP - PCR Purification beads&#160;</td>
			<td>Beckman-Coulter</td>
			<td>A63880</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynabeads&#174; M-270 Streptavidin</td>
			<td>Life Technologies</td>
			<td>65305</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">COT Human DNA, Fluorometric Grade, 1mg</td>
			<td>Roche Applied Science</td>
			<td>05480647001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit&#174; Assay Tubes&#160;</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit&#174; dsDNA HS Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
			<td></td>
		</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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit&#174; 2.0 Fluorometer&#160;</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SuperScript II Reverse Transcription 200U/ul</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D1000 ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt RNAClean XP -40ml</td>
			<td>Beckman Coulter Inc</td>
			<td>A63987</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA ScreenTape Ladder</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5578</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA ScreenTape Sample Buffer</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5577</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>Sigma</td>
			<td align="left">72068-100ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DynaBeads MyOne Streptavidin T1</td>
			<td>Life Technologies</td>
			<td>65602</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DYNAMAG -96 SIDE EACH</td>
			<td>Life Technologies</td>
			<td>12331D</td>
			<td></td>
		</tr>
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			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432-1L</td>
			<td></td>
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			<td height="20" style="height:20px;">KAPA HotStart ReadyMix</td>
			<td>KAPA Biosystems</td>
			<td>KK2602</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:407px;">My Block Mini Dry Bath</td>
			<td style="width:153px;">Benchmark</td>
			<td>BSH200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D1000 Reagents</td>
			<td style="width:153px;">Agilent Technol. Inc.</td>
			<td>5067- 5583</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:407px;">Vacufuge Plus</td>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">022829861&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted rna sequencing assay to characterize gene expression and genomic alterations

RNA-Sequenzierung (RNA-Seq) ist ein vielseitiges Verfahren, die verwendet werden können, um die Genexpression, Mutationen Genfusionen und nichtkodierenden RNAs nachzuweisen und zu charakterisieren. Standard-RNA-Seq erfordert 30-100000000 Sequenzierung liest und können mehrere RNA-Produkte wie mRNA und nicht-kodierenden RNAs enthalten. Wir zeigen, wie gezielte RNA-Seq (Capture) ermöglicht eine gezielte Untersuchung an ausgewählten RNA-Produkte einen Desktop-Sequenzer. RNA-Seq-Capture können unkommentierte, niedrig, charakterisieren oder transient Transkripte ausgedrückt, die sonst übersehen werden traditionelle RNA-Seq-Methoden. Hier beschreiben wir die Extraktion von RNA aus Zelllinien, ribosomale RNA Verarmungs, cDNA-Synthese, Herstellung von barcodierten Bibliotheken, Hybridisierung und Einfangen von Ziel Transkripte und multiplex-Sequenzierung auf einem Desktop-Sequenzer. Wir skizzieren auch die rechnerische Analyse-Pipeline, die Bewertung der Qualitätskontrolle beinhaltet, Ausrichtung, Fusion Erkennung, Genexpression Quantifizierung und Identifizierung von einzelnen Nukleotide Varianten. Dieser Test ermöglicht die gezielte Sequenzierung Transkript Genexpression, Genfusionen und Mutationen zu charakterisieren.

Eine schematische Hervorhebung Schlüsselschritte in RNA-Seq - Capture ist in Fig . 1 vier Krebszelllinien mit bekannten Mutationen , die Wirksamkeit der RNA-Seq - Capture - Technik (K562 mit ABL1 Fusion, LC2 mit RET - Fusion, EOL1 mit PDGFRalpha Fusion und RT- verwendet wurde gezeigt , demonstrieren 4 mit FGFR3 Fusion). Die vier Proben wurden gepoolt und sequenziert mit 2x 100 bp auf einem Desktop-Sequenzer liest, die fastq Dateien erzeugt. Fastq Dateien wurden durch eine RNA-Seq Analyse Pipeline laufen, die fünf Hauptkomponenten umfasst: 1) Bewertung der Qualitätskontrolle, 2) die Ausrichtung auf die menschliche Transkriptom, 3) Genexpression Quantifizierung, 4) Fusion Berufung und 5) Variante Berufung. Die Ausrichtung Datei (BAM) wird verwendet, Einzel-Nukleotid-Varianten zu nennen und die Genexpression zu berechnen. Fusions verwenden Fusion Anrufer genannt, wie TopHat Fusion (Durchführung ihrer eigenen Ausrichtung) und der Ausgang wird usin kommentierteg Fusion Erkennungssoftware. Vergleich der Genexpression von RNA-Seq und Erfassung zeigt die Anreicherung von gezielten Transkripte von 10 bis 1000-fache des Capture - Methode unter Verwendung von (2A). Zusätzlich zeigt Figur 2B eine Zunahme in dem Prozentsatz der Abbildung auf die Zielregionen Transkript liest Capture mit im Vergleich zu RNA-Seq. Bewertung der Kontrollmaßnahmen Qualität ist in Abbildung 3 dargestellt. Erfassen und RNA-Seq führen ebenso in Bezug auf die Ausrichtung auf das Transkriptom (3A, 94% vs. 93%) und Insert - Größe (3B, 174 bp vs. 162 bp) bedeuten. Verwendung des Erfassungsverfahren, ein höherer Prozentsatz an exonische Regionen sequenziert (3C, 77% vs. 60%), und umgekehrt ein geringerer Prozentsatz der Intronregionen sind sequenziert (3D, 4% vs. 20%). Insgesamt Lese Zählungen pro Probe werden in 3E dargestellt </strOng&gt;, und wie erwartet, erzeugt RNA-Seq mehr als 50-fach liest mehr als Capture. Schließlich war der Prozentsatz der rRNA - Sequenzen , die in jeder Probe niedriger mit dem Capture - Methode im Vergleich zu RNA-Seq (3F, 4% vs. 15%). Fusionserfassungsausgabe in Tabelle 1 gezeigt wird erzeugt mit normalisierten Fusionsstütz liest. Capture-RNA-Seq erfolgreich war in Fusionen für alle vier Zelllinien zu erkennen. Vergleich von Einzel - Nukleotid - Varianten genannt in überlappenden Bereichen der Aufnahme und RNA-Seq in Abbildung 4 dargestellt. Dies zeigt eine hohe Übereinstimmung der Varianten zwischen Erfassung und RNA-Seq innerhalb der Zielregion. <img alt="Abbildung 1" src="/files/ftp_upload/54090/54090fig1.jpg" /> Abbildung 1. Schematische Darstellung der RNA-Seq - Capture - Schritte. In dieser experimentellen Demonstration ist RNA erste depleted ribosomaler RNA, die durch chemische Fragmentierung und Synthese von komplementärer DNA (cDNA) unter Verwendung von reverser Transkriptase, gefolgt. Als nächstes wird die cDNA polyadenylierte und an beiden Enden an plattformspezifischen Adapter ligiert, um eine Bibliothek zu erzeugen. Nur cDNA-Bibliotheken mit der richtigen Adapter werden dann durch PCR amplifiziert. Bibliotheken werden dann an individuelle Oligonukleotid-Sonden hybridisiert und unter Verwendung von magnetischen Kügelchen eingefangen. Diese geringe Menge der aufgenommenen Bibliothek muss ein zweites Mal für die nächste Generation Sequenzierung genug, um verstärkt werden. Mehrere Bibliotheken können dann parallel sequenziert werden. Die Sequenzierung Daten werden für die RNA - Ereignisse von Interesse, wie Genfusionen, Expression oder Mutationen analysiert. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54090/54090fig2.jpg" /> Abbildung 2 CERGLEICH von Targeted Gene im Aufnahme gegen RNA-Seq. A, Genexpression Vergleich zwischen Erfassung und RNA-Seq in vier Krebszelllinien K562, LC2, EOL1 und RT-4 gemessen liest pro Kilobasen- pro Million abgebildet liest (FPKM) (Log - Skala). Gezielte Gene von Interesse angereichert sind (blau) im Vergleich zu Nicht-Zielgene (grau). B, Prozentsatz von Reads Zuordnung zu Zielregion erhöht wird im Aufnahme gegen RNA-Seq - Bibliotheken in vier Krebszelllinien. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig2large.jpg" target="_blank">um eine größere Version diese Figur.</a> <img alt="Figur 3" src="/files/ftp_upload/54090/54090fig3.jpg" /> Abbildung 3. Die Sequenzierung Metrics von Capture im Vergleich zu RNA-Seq in vier repräsentativen Krebszelllinien. A, Prozentsatz der Abbildung auf die Transkriptom liest, <strong&gt; B, Insertgröße von Bibliotheken bedeuten. C, Prozentsatz der in exonische Regionen liest. D, Prozentsatz der in Intron Regionen liest. E, Totalsequenzierung liest. F, Prozentsatz der Abbildung auf der ribosomalen RNA liest. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig3large.jpg" target="_blank">Bitte klicken Sie hier anzuschauen größere Version der Figur.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> Zelllinie </td><td rowspan="2"> Verschmelzung </td><td rowspan="2"> Bibliothek Typ </td><td rowspan="2"> Gesamt gelesen </td><td rowspan="2"> On Target Liest </td><td colspan="3"> Normierte Fusion Unterstützung Liest (NFSR) </td></tr><tr><td> TophatFusion </td><td> ChimeraScan </td><td> TROBEN </td></tr><tr><td> K562 </td><td> BCR-ABL </td><td> RNA-Seq </td><td> 150300482 </td><td> 279438 </td><td> 0 </td><td> 438 </td><td> 0 </td></tr><tr><td></td><td></td><td> Erfassung </td><td> 9341148 </td><td> 7566087 </td><td> 598 </td><td> 343 </td><td> 0 </td></tr><tr><td> LC2 </td><td> CCDC6-RET </td><td> RNA-Seq </td><td> 128861790 </td><td> 307566 </td><td> 0 </td><td> 97 </td><td> 0 </td></tr><tr><td></td><td></td><td> Erfassung </td><td> 12320692 </td><td> 10314284 </td><td> 71 </td><td> 44 </td><td> 6 </td></tr><tr><td> EOL1 </td><td> FIP1L1-PDGFRA </td><td> RNA-Seq </td><td> 135321406 </td><td> 225222 </td><td> 0 </td><td> 0 </td><td> 170 </td></tr><tr><td></td><td></td><td> Erfassung </td><td> 9317418 </td><td> 7680818 </td><td> 143 </td><td> 0 </td><td> 7 </td></tr><tr><td> RT4 </td><td> FGFR3<html> -TACC3 </td><td> RNA-Seq </td><td> 161350024 </td><td> 208741 </td><td> 0 </td><td> 131 </td><td> 469 </td></tr><tr><td></td><td></td><td> Erfassung </td><td> 8305950 </td><td> 6563574 </td><td> 358 </td><td> 88 </td><td> 34 </td></tr></tbody></table> Tabelle 1. Fusion Erkennung für Capture im Vergleich zu RNA-Seq von K562, LC2, EOL1 und RT-4. Diese Tabelle zeigt vier Krebszelllinien und drei verschiedenen Fusionserkennungsalgorithmen, TopHat2, ChimeraScan und Trup in dieser Demonstration genutzt. Diese Tabelle zeigt die Fähigkeit, Fusionen mit Capture zu erkennen, die weniger als insgesamt 10 Millionen liest im Vergleich zu mehr als 60 Millionen liest für RNA-Seq genutzt. Fusion Stütz liest berechnet wurden durch Fusion Dividieren Unterstützung liest von Kinase liest, multipliziert mit einer Million. <img alt="&lt;/ Html">&quot;4&quot; src = &quot;/ files / ftp_upload / 54090 / 54090fig4.jpg&quot; /&gt; Abbildung 4. SNV Aufruf für Capture im Vergleich zu RNA-Seq. Diese Venn - Diagramme zeigen die Anzahl der Single Nucleotide Varianten (SNVs) , die durch Abscheidung und RNA-Seq für jede der vier Zelllinien (K562, LC2, EOL1 und RT-4) nachgewiesen wurden. Dies zeigt hohe Übereinstimmung von SNVs zwischen Capture and RNA-Seq innerhalb gezielte-Region. K562 (81,3%), LC2 (78,3%), EOL1 (89,5%) und RT-4 (73,9%) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig4large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig4large.jpg" target="_blank">eine größere Version zu sehen diese Figur.</a>

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.

RNA-Sequenzierung Assay Gezieltes Genexpression und Genomic Änderungen an Charakterisieren

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

targeted-rna-sequencing-assay-to-characterize-gene-expression-genomic

Nanyang Technological University

Research

JoVE Journal

Biology

10.3K Aufrufe.  The Ohio State University. 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.

Serie: Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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

Forschung

Biologie

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

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Paraffin-Embedded-und Gefrierschnitte von Drosophila Adult Muskeln

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

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

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

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

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Erkennen Somatische Genetische Veränderungen in Tumorproben von Exon-Abscheidung und-Massively Parallel Sequencing

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

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

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

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.