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.

RNAseq 캡처 RNAseq 및 마이크로 어레이 사이의 중간 전략은 사체의 선택된 부분을 평가하기위한 접근이다. 캡처의 이점은 유전자 변형의 데스크탑 시퀀서, 높은 처리량, 및 검출에 선정 신속한 처리 시간 감소를 포함한다. 이 방법은, 비 코딩 RNA를 (23)의 특성을 단일 염기를 검출하도록 구성 될 수있는 RNA의 스 플라이 싱을 조사하고 유전자 융합체 또는 구조적 재 배열 (24)을 식별하기 위해, 4-6 변종. 또한,이 방법은 파라핀 블록 (24, 25)에 포르말린으로 고정 후의 임베디드 한 임상 샘플 또는 처리에 적용될 수있다. 마이크로 어레이에 비해 RNAseq 캡처 몇 가지 중요한 이점이 있으며, 실시간 정량 PCR, 생거 시퀀싱 및 DNA 시퀀싱. 마이크로 어레이 인해 크로스 하이브리드 및 프로브의 결합이 아닌 특정 높은 배경에 의해 제한된다. 리터와 유전자의 정량고도로 발현 유전자 측정 한 채도 신호의 영향을받는 동안 흐름 식은, 배경 노이즈로 인해 제한된다. RNAseq 캡처 비교, 실시간 PCR 재현하기 어려운 증명한다. 또한, RNAseq이 새로운 성적 증명서의 검출을 허용, 이하 시작 입력 자료를 필요로하며, 생거 시퀀싱 대안 스 플라이 싱 (26) .IN 대비를 검색 할 수 있습니다, RNAseq는 높은 처리량과 낮은 표현의 miRNA의 분석을 할 수 있습니다. 생거 시퀀싱 그러나 새로운 융합의 식별이 선험적 후보 중단의 요구에 의해 방해 알려진 엑손 - 엑​​손 접합 및 체세포 DNA 돌연변이와 융합의 검증을위한 유용한 도구로 입증했다. 비용 효율적인되지 DNA 시퀀싱, 데이터의 큰 저장 공간을 필요로하고, 전사 후 변형 검출 불가능하다. RNAseq 캡처에 관련된 몇 가지 중요한 단계가 있습니다. 우선, 도서관에서의 제품 수율을 향상시킬세척시 RNA / cDNA를 특정 상자성 구슬과 상자성 비즈, 이상 수율의 손실로 이어질 것입니다 구슬을, 건조하지 않도록주의해야합니다. 또한,하지의 밑에 건조 된 cDNA 수율을 감소시킬 수 에탄올, 모든 에탄올 샘플 튜브에서 제거되어 있는지 확인 구슬을. 둘째, 상호 보완적인 프로브의 cDNA 라이브러리의 하이브리드 우리가 사전에 적어도 두 시간 동안 65 ° C로 온난화 워시 버퍼 I과 엄격한 버퍼를 추천 일관된 온도에 따라 달라집니다. 또한, 하이브 리다이 제이션 이후의 결합 및 세척 단계 동안 65 ° C를 유지하기 위해 필수적이다. 여기에 사용되는 프로브는 키나아제, 같은 전사 인자, 집 유지 유전자와 같은 일반 재배에 관여하는 유전자를 포함하는 약물 개발에 대한 관심의 유전자의 엑손을 위해 설계되었다. 또한, 유전자 함량은 사용자 정의 및 캡처 패널 크기는 다를 수 있습니다. 게놈 영역에 새로운 정보가 발생로서 또한, 추가적인 프로브 설계와 T에 추가 될 수있다그는 캡처 패널을 기존. 정렬 측정 평가, 특히 온 - 목표 비율은 대상 영역이 강화되었다 얼마나 잘에 대한 정보를 제공합니다. 낮은 온 - 목표 속도는 원하는 대상 영역을 캡처 및 농축되지 않은 이에 실패 하이브리드 캡처, 때문일 수 있습니다. 이 경우, 라이브러리 세트의 재 혼성화 캡처가 수행되어야한다. 낮은 온 - 레이트 목표는 또한 샘플의 rRNA의 비율을 산출함으로써 확인할 수있다 rRNA의 고갈 실패로 될 수있다. 샘플 내의 rRNA의 높은 비율은 rRNA의 고갈로 시작하는 샘플의 재 조제를 필요로한다. 라이브러리 농도 혼성화 캡처위한 요구 이하로 떨어질 경우, 또한, 상기 샘플 종류와 품질 (: 50 ~ 1,000 ng의 범위)에 대해 시작 입력의 양을 최적화하는 것이 바람직 할 것이다. 대상 RNAseq 응용 프로그램에 대한 몇 가지 장점이 있지만, 또한에 제한 사항이 있습니다중히 여기다. 가난한 RNA의 RIN에 따라 품질이나 분열의 정도와 샘플 시퀀싱 품질 라이브러리를 생성 할 수 없습니다. 여러 그룹이 포르말린 고정 파라핀 포함 된 샘플의 성공을 증명하고있다, 그러나 순서 24,25,27에 대한 통과하지 않습니다 샘플이 있습니다. RNAseq 캡처 알려진 성적 증명서에 초점을 맞추고 있기 때문에, 그것은 소설이나 주석이 달려 있지 않은 성적에 대한 편견 RNAseq의 장점을 잃는다. 또한, SNP 검출을위한 방법 RNAseq는 표현 사체의 돌연변이를 검출 할 수있다. RNAseq 캡처의 미래의 기회는 연구와 임상 응용 프로그램을 포함한다. 긴 비 코딩 RNA의 수천 생물학에서의 역할의 최근 발견은 초점을 맞춘 특성화가 필요합니다. 암, 감염성 질환, 및 비 침습적 검사로 인간 질병의 특성을 병원에서 RNAseq 캡처 연구 테스트를 넘어 확장 할 수 있으며, 임상 분석으로 번역합니다. 게놈 서열과 함께 approaCHES, RNAseq 캡처 연구하고 표현 게놈의 특성을 통합 할 수 있습니다.

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>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<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 서열 (RNAseq)는 유전자 발현 돌연변이 유전자 융합체, 및 비 암호화 RNA를 검출하고 특성화하는 데에 이용 될 수있는 다목적 방법이다. 1 억 순서 읽고 등의 mRNA 및 비 암호화 RNA를 여러 RNA 제품을 포함 할 수있다 - 표준 RNAseq (30)가 필요합니다. 우리는 타겟 RNAseq (캡처) 바탕 화면 시퀀서를 사용하여 선택 RNA 제품에 초점을 맞춘 연구를 허용하는 방법을 보여줍니다. RNAseq 캡처 그렇지 않으면 기존의 RNAseq 방법을 사용하여 놓칠 수있다, 주석이 달려 있지 않은 저, 또는 일시적으로 표현 된 성적 증명서를 특성화 할 수 있습니다. 여기에서 우리는 세포 라인, 리보솜 RNA 고갈, cDNA 합성, 바코드 라이브러리, 바탕 화면 시퀀서에 하이브리드 및 목표 성적 증명서의 캡처 및 다중 서열의 준비에서 RNA의 추출을 설명합니다. 또한 품질 관리 평가, 정렬 검출 융합 유전자 발현의 정량화 및 단일 NUC의 식별을 포함하는 연산 파이프 라인을 분석, 개요leotide 변형. 이 분석은 유전자 발현, 유전자 융합, 돌연변이의 특성을 타겟팅 사체 서열을 허용한다.

RNAseq 캡처 개략적 강조 주요 단계가도 1에 도시되어있다. 공지 된 돌연변이와 함께 4 개의 암 세포주는 RNAseq 캡처 기술의 유효성을 증명 하였다 (K562 ABL1 융합, RET 융합과 LC2와, EOL1 PDGFRalpha 융합 및 RT-으로 4 FGFR3 융합과). 네 개의 샘플을 함께 모으고 100 혈압 FASTQ 파일을 생성하는 데스크탑 시퀀서에 판독 2 배속으로 서열화 하였다. 인간 사체 1) 품질 관리 평가 2) 정렬 3) 유전자 발현의 정량 분석​​, 4) 융합 호출 및 5) 변형 호출 : FASTQ 파일 다섯 가지 주요 구성 요소를 포함하는 RNAseq 분석 파이프 라인을 통해 실행 하였다. 정렬 파일 (BAM)은 단일 염기 변이를 호출 유전자 발현을 계산하는데 사용된다. 융합은 (자신의 정렬 수행) 등 TopHat 융합 등 융합 발신자를 사용하여 호출되고, 출력은 날기를 주석이g 융합 탐지 소프트웨어. 유전자 RNAseq에서 표현과 캡처의 비교 (10)에 의해 표적 성적 증명서의 농축을 보여줍니다 캡처 방법 (그림 2A)를 사용하여 1,000 배. RNAseq 비교 포착하여 타겟 사체 영역에 매핑 된 읽기 또한,도 2b는 퍼센트의 증가를 나타낸다. 품질 관리 대책의 평가는 그림 3에 표시됩니다. 캡처 RNAseq은 사체 (3A, 94 % 대 93 %)에 정렬의 관점에서 동일하게 수행하고 삽입 크기 (3B, 174 bp의 대 (162) BP)를 의미한다. 캡쳐 방법을 사용하여, 엑손 영역의 높은 비율은 서열화되어있다 (3C 77 % 대 60 %), 반대로 인트론 영역의 하부 비율 서열화된다 (3D, 4 % 대 20 %). 샘플 당 총 읽기 수는 3E에 도시된다 </str예상대로 50 배 더 캡처보다 읽기를 통해 사연&gt;, 그리고 RNAseq 생성. 마지막으로, 각 샘플에 존재 rRNA의 서열의 비율 RNAseq (3 층, 4 % 대 15 %)에 비해 포획 방법을 이용하여 낮았다. 표 1에 나타낸 용융 검출 출력은 표준화 된 융합 판독지지 생성된다. RNAseq 4 개의 모든 세포주 융합체 검출에 성공 캡처. 캡처 및 RNAseq의 영역을 중첩라는 단일 염기 변이의 비교는 그림 4에 표시됩니다.이 대상 영역 내에서 캡처 및 RNAseq 사이 변종의 높은 일치도를 보여줍니다. <img alt="그림 1" src="/files/ftp_upload/54090/54090fig1.jpg" /> 그림 1. 도식 RNAseq 캡처 단계의.이 실험 데모에서는, RNA가 먼저 deple입니다역전사 효소를 사용하여 화학적 분열 보완 DNA (cDNA의)의 합성 다음 리보솜 RNA의 테드. 다음으로, cDNA를 폴리아 데 닐화 및 라이브러리를 생성하기 위해 플랫폼 별 어댑터 양단에 결찰된다. 적절한 어댑터 만의 cDNA 라이브러리는 다음 PCR에 의해 증폭된다. 라이브러리는 사용자 정의 올리고 뉴클레오티드 프로브에 하이브리드 및 자기 구슬을 사용하여 캡처됩니다. 캡처 라이브러리이 소량 차세대 시퀀싱 충분를 한 번 증폭되어야한다. 다중 라이브러리는 병렬로 순서화 될 수있다. 시퀀싱 데이터는 유전자 융합, 발현 또는 돌연변이 같은 관심의 RNA 이벤트를 분석한다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54090/54090fig2.jpg" /> 그림 2. CRNAseq. A, 측정 네 개의 암 세포 라인 K562, LC2, EOL1 및 RT-4 캡처 및 RNAseq 사이의 유전자 발현 비교 대 캡처에서 표적 유전자의 omparison 매핑 만 읽고 (FPKM) (로그 스케일) 당 킬로 당 읽습니다. 관심의 표적 유전자가 아닌 타겟 유전자 (회색). B, 대상 영역에 매핑이 네 가지 암 세포주에서 RNAseq 라이브러리 대 캡처에 증가 읽기의 비율 비교 (파란색) 강화됩니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig2large.jpg" target="_blank">여기를 클릭하십시오이의 더 큰 버전을 볼 수 있습니다 이 그림.</a> <img alt="그림 3" src="/files/ftp_upload/54090/54090fig3.jpg" /> 네 개의 대표 암 세포주에서 RNAseq 대 캡처. A는, 비율로는 사체에 매핑을 읽고 시퀀싱 메트릭 그림 3, <stroNG&gt; B가, 라이브러리 삽입 크기를 의미한다. C를,의 비율은 엑손 지역에서 읽습니다. D의 비율이 인트론 지역에서 읽습니다. E를, 총 순서가. F, 리보솜 RNA에 매핑을 읽는 비율이 읽습니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig3large.jpg" target="_blank">을 보려면 여기를 클릭하십시오 이 그림의 더 큰 버전.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 세포주 </td><td rowspan="2"> 퓨전 </td><td rowspan="2"> 도서관 유형 </td><td rowspan="2"> 총 읽어 </td><td rowspan="2"> 대상 읽고에 </td><td colspan="3"> 지원 정규화 퓨전 읽고 (NFSR) </td></tr><tr><td> TophatFusion </td><td> ChimeraScan </td><td> TR쪽으로 </td></tr><tr><td> K562 </td><td> BCR-ABL </td><td> RNAseq </td><td> 150300482 </td><td> 279438 </td><td> 0 </td><td> (438) </td><td> 0 </td></tr><tr><td></td><td></td><td> 포착 </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> RNAseq </td><td> 128861790 </td><td> 307566 </td><td> 0 </td><td> 97 </td><td> 0 </td></tr><tr><td></td><td></td><td> 포착 </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> RNAseq </td><td> 135321406 </td><td> 225222 </td><td> 0 </td><td> 0 </td><td> (170) </td></tr><tr><td></td><td></td><td> 포착 </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> RNAseq </td><td> 161350024 </td><td> 208741 </td><td> 0 </td><td> (131) </td><td> 469 </td></tr><tr><td></td><td></td><td> 포착 </td><td> 8305950 </td><td> 6563574 </td><td> (358) </td><td> (88) </td><td> (34) </td></tr></tbody></table> K562, LC2, EOL1 및 RT-4의 RNAseq 대 캡처 표 1. 퓨전 감지.이 표는이 데모에 사용되는 네 개의 암 세포 라인과 세 가지 융합 탐지 알고리즘, TopHat2, ChimeraScan 및 TRUP를 표시합니다. 이 표는보다 큰 60000000가 RNAseq 활용 읽기에 비해 읽기 미만 1,000 만 총을 사용하여 캡처와 융합을 감지 할 수있는 능력을 보여줍니다. 키나아제 판독하여 융착지지 융합 나누어 계산 하였다 판독지지 1 백만 곱 읽는다. <img alt="&lt;/ HTML">&quot;그림 4&quot;SRC = &quot;/ 파일 / ftp_upload / 54090 / 54090fig4.jpg&quot;/&gt; RNAseq 대 캡처를 요구 그림 4. SNV.이 벤 다이어그램은 네 개의 세포주의 각각에 대해 캡처 및 RNAseq에 의해 검출 된 단일 염기 변형 (SNVs)의 수를 표시 (K562, LC2, EOL1 및 RT-4). 이 높은 대상 영역 내에서 캡처 및 RNAseq 사이 SNVs의 일치 보여줍니다. K562 (81.3 %), LC2 (78.3 %), EOL1 (89.5 %)과 RT-4 (73.9 %)을 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54090/54090fig4large.jpg" target="_blank">의 더 큰 버전을 보려면 여기를 클릭하십시오 이 그림.</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 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로

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 조회수.  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.

비디오: 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.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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

연구

생물학

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.

유전자 발현 분석을위한 배아 Zebrafish 및 cDNA 합성에서 RNA 격리

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.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

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.

활성 T.의 체외의 Reconstitution에서 castaneum의 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.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

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.

엑손 캡처 및 대규모 병렬 염기 서열 종양 검체에서 체세포 유전 변경을 검출

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.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

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.