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首页> 外文期刊>Journal of biomolecular techniques :JBT. >High-Throughput Strand-Specific mRNA Library Preparation for Illumina Sequencing from Total RNA Isolated from Normal and Cancerous Ovary Tissue
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High-Throughput Strand-Specific mRNA Library Preparation for Illumina Sequencing from Total RNA Isolated from Normal and Cancerous Ovary Tissue

机译：高通量链特异性mRNA文库制备，用于从正常和癌性卵巢组织中分离的总RNA进行Illumina测序

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摘要

The cost benefits of whole genome and transcriptome next generation sequencing (NGS) have revolutionized genetic analysis. Determination of regulatory changes in cancer cells by whole transcriptome analysis has proven useful for tailoring treatment options for patients. An efficient, consistent and reliable whole transcriptome library preparation method is necessary for successful transition of the NGS technologies from research to clinical applications. Due to the labile nature of mRNA and the tedium of processing large numbers of patient samples, automation would greatly improve the reliability and throughput of whole transcriptome library preparation. We have explored the automation of whole transcriptome library preparation from total RNA of normal and cancerous ovary tissue. The Apollo 324? System (IntegenX Inc.) was used to isolate poly A mRNA from 6 to 48 samples of total RNA and prepare strand-specific mRNA libraries for sequencing on the Genome Analyzer IIx (Illumina). The cDNA output was amplified on a bench thermocycler to yield whole transcriptome libraries in a single eight hour day. While most conventional RNA-Seq library preparation methods convert mRNA to cDNA, in our strand specific library preparation, we ligated the adapters directly to fragmented mRNA to preserve strand polarity. Preserving strand polarity of the transcript reduces the bioinformatics bottleneck. A commercially available manual strand-specific library was used as the bench control for comparison and validation of our library preparations. The gene expression profile of 12 up-regulated and down-regulated genes was equivalent between the high-throughput libraries and the published data. The isolated polyA mRNA had an average of 0.3% rRNA contamination from 500 ng of total RNA. There was a 75% time reduction for automated library preparation from total RNA compared with conventional library preparation methods. There was 90% correlation of gene expression between the automated and bench library preparation methods for up to 48 samples. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.
机译：全基因组和转录组下一代测序（NGS）的成本优势已经彻底改变了遗传分析。通过全转录组分析确定癌细胞中的调节变化已被证明可用于定制患者的治疗选择。有效，一致和可靠的整个转录组文库制备方法对于NGS技术从研究到临床应用的成功过渡是必要的。由于mRNA的不稳定特性和处理大量患者样品的繁琐工作，自动化将大大提高整个转录组文库制备的可靠性和通量。我们已经探索了从正常和癌性卵巢组织的总RNA中提取整个转录组文库的自动化方法。阿波罗324？系统（IntegenX Inc.）用于从6到48个总RNA样品中分离poly A mRNA，并准备用于在Genome Analyzer IIx（Illumina）上测序的链特异性mRNA文库。 cDNA输出在台式热循环仪上扩增，可在单天的八小时内产生完整的转录组文库。尽管大多数常规RNA-Seq文库制备方法将mRNA转换为cDNA，但在我们的链特异性文库制备中，我们将衔接子直接连接至片段化的mRNA，以保留链极性。保留转录本的链极性可减少生物信息学瓶颈。使用市售的手动链特异性文库作为基准对照，用于比较和验证我们的文库制备物。在高通量文库和已发表的数据之间，12个上调和下调的基因的基因表达谱是等效的。从500 ng总RNA中分离出的polyA mRNA平均有0.3％rRNA污染。与常规文库制备方法相比，从总RNA进行自动文库制备的时间减少了75％。自动和台式文库制备方法之间的基因表达有90％的相关性，最多可处理48个样品。这里由生物分子资源设施协会提供了《生物分子技术杂志》上的文章：JBT。

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