Ribosomal RNA Depletion for Massively Parallel Bacterial RNA-Sequencing Applications
Ribosomal RNA Depletion for Massively Parallel Bacterial RNA-Sequencing Applications
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DOI：
10.1007/978-1-61779-089-8_7
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Duan, Xiaoping
中科院分区：
文献类型：
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作者：
Chen, Zhoutao;Duan, Xiaoping
rna测序（RNA-Seq）是使用下一代测序技术的转录组数字显示，并提供转录组的详细，高通量视图。RNA- seq的主要步骤是从总RNA中分离整个转录组。由于大核糖体RNA （large ribosomal RNA, rRNA）约占总RNA的90%，使用现有的RNA分离方法很难进行不受rRNA污染的全转录组分析。RiboMinus （TM）纯化方法为从总RNA中分离不含大rRNA的转录组RNA分子进行转录组分析提供了一种新颖有效的方法。它允许通过从总RNA中选择性地消耗丰富的rRNA分子来分离整个转录组。rRNA缺失的RNA片段被称为RiboMinus (TM) RNA片段，它富含聚腺苷化RNA、非聚腺苷化RNA、预处理RNA、tRNA、许多调控RNA分子和其他功能未知的RNA转录物。采用RiboMinus （TM）方法分离RiboMinus RNA，生物分析仪分析结果显示，从0.5 ~ 10 μ g细菌总RNA中，165和23S rRNA分子去除率高达99.0%。它可以实现高效的全转录组测序分析，而不会受到高度丰富的rRNA的严重污染。根据SOLD和Genome Analyzer RNA-Seq数据，残余rRNA占整个转录组的不到10%。
RNA-sequencing (RNA-Seq) is a digital display of a transcriptome using next-generation sequencing technologies and provides detailed, high-throughput view of the transcriptome. The lint step in RNA-Seq is to isolate whole transcriptome from total RNA. Since large ribosomal RNA (rRNA) constitutes approximately 90% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult using existing RNA isolation methods. RiboMinus (TM) purification method provides a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large rRNA from total RNA for transcriptome analysis. It allows for whole transcriptome isolation through selective depletion of abundant rRNA molecules from total RNA. The rRNA depleted RNA fraction is termed as RiboMinus (TM) RNA fraction, which is enriched in polyadenylated RNA, nonpolyadenylated RNA, preprocessed RNA, tRNA, numerous regulatory RNA molecules, and other RNA transcripts of yet unknown function. Using RiboMinus (TM) method to isolate RiboMinus RNA results in up to 99.0% removal of 165 and 23S rRNA molecules from 0.5 to 10 mu g total bacterial RNA based on Bioanalyzer analysis. It enables efficient whole transcriptome sequencing analysis without major contamination from highly abundant rRNA. Residual rRNA accounts for less than 10% of entire transcriptome based on both SOLD and Genome Analyzer RNA-Seq data.