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.

注意：此协议说明了同时处理和4个样品的分析。这种方法是用RNA从细胞中分离，新鲜冷冻组织和福尔马林固定，石蜡包埋的组织的（FFPE）兼容。该协议始于50 - 从RNA输入每个样本的1000纳克（250纳克推荐）。 1. rRNA的消耗和RNA议事碎片<ol><li> rRNA的消耗 <ol><li>在室温下除去洗脱，素，片段组合，rRNA的去除组合，rRNA的结合缓冲液和重悬浮缓冲液从-20℃并解冻。从4?...

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat Rev Genet. 10, 57-63 (2009).</li><li>Mercer, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sequencing+for+gene+discovery+and+quantification+using+RNA+CaptureSeq.">Targeted sequencing for gene discovery and quantification using RNA CaptureSeq.</a> Nat Protoc. 9, 989-1009 (2014).</li><li>Maher, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+transcript+discovery+by+paired-end+transcriptome+sequencing.">Chimeric transcript discovery by paired-end transcriptome sequencing.</a> Proc Natl Acad Sci U S A. 106, 12353-12358 (2009).</li><li>Piskol, R., Ramaswami, G., Li, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+identification+of+genomic+variants+from+RNA-seq+data.">Reliable identification of genomic variants from RNA-seq data.</a> Am J Hum Genet. 93, 641-651 (2013).</li><li>Quinn, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+strategies+for+SNP+detection+in+RNA-seq+data:+application+to+lymphoblastoid+cell+lines+and+evaluation+using+1000+Genomes+data.">Development of strategies for SNP detection in RNA-seq data: application to lymphoblastoid cell lines and evaluation using 1000 Genomes data.</a> PLoS One. 8, e58815 (2013).</li><li>Tang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eSNV-detect:+a+computational+system+to+identify+expressed+single+nucleotide+variants+from+transcriptome+sequencing+data.">The eSNV-detect: a computational system to identify expressed single nucleotide variants from transcriptome sequencing data.</a> Nucleic Acids Res. 42, e172 (2014).</li><li>. Preparing Libraries for Sequencing on the MiSeq&#174; Available from: <a target="_blank" href="https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/preparing-libraries-for-sequencing-on-miseq-15039740-d.pdf">https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/preparing-libraries-for-sequencing-on-miseq-15039740-d.pdf</a> (2013)</li><li>. MiSeq&#174; Reagent Kit v2 Reagen Preparation Guide Available from: <a target="_blank" href="https://support.illumina.com/downloads/miseq_reagent_kit_reagent_preparation_guide.html">https://support.illumina.com/downloads/miseq_reagent_kit_reagent_preparation_guide.html</a> (2012)</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biol. 14, R36 (2013).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>DeLuca, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-SeQC:+RNA-seq+metrics+for+quality+control+and+process+optimization.">RNA-SeQC: RNA-seq metrics for quality control and process optimization.</a> Bioinformatics. 28, 1530-1532 (2012).</li><li>Dobin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAR:+ultrafast+universal+RNA-seq+aligner.">STAR: ultrafast universal RNA-seq aligner.</a> Bioinformatics. 29, 15-21 (2013).</li><li>Van der Auwera, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+FastQ+data+to+high+confidence+variant+calls:+the+Genome+Analysis+Toolkit+best+practices+pipeline.">From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.</a> Curr Protoc Bioinformatics. 11, 11.10.1-11.10.33 (2013).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat Biotechnol. 28, 511-515 (2010).</li><li>Iyer, M. K., Chinnaiyan, A. M., Maher, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChimeraScan:+a+tool+for+identifying+chimeric+transcription+in+sequencing+data.">ChimeraScan: a tool for identifying chimeric transcription in sequencing data.</a> Bioinformatics. 27, 2903-2904 (2011).</li><li>Kim, D., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat-Fusion:+an+algorithm+for+discovery+of+novel+fusion+transcripts.">TopHat-Fusion: an algorithm for discovery of novel fusion transcripts.</a> Genome Biol. 12, R72 (2011).</li><li>Fernandez-Cuesta, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+fusion+genes+in+lung+cancer+using+breakpoint+assembly+of+transcriptome+sequencing+data.">Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data.</a> Genome Biol. 16, 7 (2015).</li><li>Shugay, M., Ortiz de Mendibil, ., Vizmanos, I., L, J., Novo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncofuse:+a+computational+framework+for+the+prediction+of+the+oncogenic+potential+of+gene+fusions.">Oncofuse: a computational framework for the prediction of the oncogenic potential of gene fusions.</a> Bioinformatics. 29, 2539-2546 (2013).</li><li>Clark, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+gene+profiling+of+long+noncoding+RNAs+with+targeted+RNA+sequencing.">Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing.</a> Nat Methods. 12, 339-342 (2015).</li><li>Cieslik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+exome+capture+RNA-seq+for+highly+degraded+RNA+with+application+to+clinical+cancer+sequencing.">The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing.</a> Genome Res. , (2015).</li><li>Cabanski, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cDNA+hybrid+capture+improves+transcriptome+analysis+on+low-input+and+archived+samples.">cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.</a> J Mol Diagn. 16, 440-451 (2014).</li><li>Costa, C., Gimenez-Capitan, A., Karachaliou, N., Rosell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+screening:+from+the+RT-PCR+to+the+RNA-seq.">Comprehensive molecular screening: from the RT-PCR to the RNA-seq.</a> Transl Lung Cancer Res. 2, 87-91 (2013).</li><li>Zhao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA-Seq+by+poly+(A)+capture,+ribosomal+RNA+depletion,+and+DNA+microarray+for+expression+profiling.">Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.</a> BMC Genomics. 15, 419 (2014).</li></ol>

RNA测序Capture是RNA测序和芯片之间的中间策略评估转录的选定部分接近。捕获的优点包括在桌面序器，高吞吐量，和基因组改变的检测降低的成本，快速的周转时间。可适于表征非编码RNA 23，检测单核苷酸的方法变体4-6，检查RNA剪接，并确定基因融合或结构重排24。此外，这种方法可应用于已经经过用福尔马林固定并包埋在石蜡块24,25临床或处理的样本。 <p class=...

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>
			<td style="width:153px;">Eppendorf</td>
			<td style="width:143px;">21516-166</td>
			<td style="width:167px;"></td>
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		<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>
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			<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>
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		<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>
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targeted rna sequencing assay to characterize gene expression and genomic alterations

RNA测序（RNA测序）是可以被用于检测和表征的基因表达，突变，基因融合，和非编码RNA一个通用的方法。标准RNA测序需要30 - 亿测序读出并可以包括多个RNA产物如mRNA和非编码RNA。我们证明有针对性的RNA测序（捕获）如何允许在使用桌面序选定的RNA产品聚焦研究。 RNA测序采集可以表征可能以其它方式使用传统的RNA测序方法错过没有注释，低或瞬时表达成绩单。在这里，我们描述了从细胞系，核糖体RNA耗尽，cDNA合成，制备条形码库，杂交和靶向转录物的捕获和多重测序上的桌面序的RNA的提取。我们也勾勒出计算分析管道，其中包括质量控制评估，校准，检测融合，基因表达定量和单国统会的鉴定leotide变种。该测定允许有针对性的转录测序来表征的基因表达，基因融合，和突变。

在RNA测序捕捉的概略突出关键步骤示于图1。与已知的突变四癌细胞系被用来证明RNA测序捕捉技术的有效性（K562与ABL1融合，LC2与RET融合，EOL1与PDGFRalpha融合和RT- 4 FGFR3融合）。这四个样品汇集在一起​​，并进行测序2倍于台式机音序器，生成FASTQ文件100个基点的读取。 1）质量控制评估，2）对准至人类转录，3）基因表达的定量，4）?...

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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.

有针对性的RNA测序分析表征基因表达和基因组改变

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

Research

JoVE Journal

Biology

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

主电穿孔的小鼠骨髓造血细胞培养制备

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

科研

生物学

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

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

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

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

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

在体外重组活跃T。谷盗端粒酶

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

条形码酵母图书馆竞争力的基因屏幕

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

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

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

全球合成的RNA在单细胞水平以下的低氧分析

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