基于单分子测序仪的基因表达分析新技术[创新技巧]

生物通报道，日本横滨市RIKEN Omics科学中心（RIKEN Omics Science Center）的研究人员开发出一种新的基因表达分析技术，只需100 ng总RNA，就能准确定量地测定基因表达水平。该成果近日发表在《Genome Research》在线版上。

这项技术称为“HeliScopeCAGE”，它将RIKEN的CAGE（Cap Analysis of Gene Expression）与第三代测序公司Helicos BioSciences的Helicos遗传分析系统相结合，为稀有细胞群体的基因表达网络分析开启了大门。

CAGE是近年出现的一种分析RNA转录本的高效方法。它由日本科学家Hayashizaki等开发，能产生样品中mRNA的5’端快照。这种技术实现了高通量的基因表达分析以及转录起点的图谱分析。

目前有很多方法能确定转录本的丰度，包括RT-PCR、EST测序、SAGE和大规模平行测序等，这其中大部分都依赖3’端的序列。但是对于转录起点和相关启动子的鉴定，5’端特异的特征序列则是表达谱注释所必需的。因此，研究人员开始利用CAGE进行5’端短序列标签的克隆。

近几年，新一代测序仪就基因在分子水平如何表达提供了日渐详细的信息。这些基因的转录产物揭示了转录本结构和功能的复杂度，为了解癌症及其他疾病的分子性质提供了线索。

对于HeliScopeCAGE，研究人员修改了原先的CAGE步骤，以便与HeliScope 单分子测序仪共同使用。与之前的测序仪不同，HeliScope测序仪不采用PCR反应来扩增DNA链，此过程可能会将偏差引入数据。相反，HeliScope测序仪真正对DNA链本身测序，实现了高度精确的测定。

RIKEN的研究人员证实了这种直接方法能减少偏差，为低至100 ng，高至5 μg的总RNA带来高度重复的数据。白血病细胞系THP-1和人子宫颈癌细胞系HeLa之间的比较进一步说明，这种技术产生的结果与传统芯片分析的结果高度相关。（生物通 余亮）

相关阅读：

如何开展CAGE和nanoCAGE

原文摘要：

Unamplified cap analysis of gene expression on a single-molecule sequencer

Published in Advance May 19, 2011, doi: 10.1101/gr.115469.110

摘要：
We report the development of a simplified cap analysis of gene expression (CAGE) protocol adapted for single-molecule sequencers that avoids second strand synthesis, ligation, digestion, and PCR. HeliScopeCAGE directly sequences the 3′ end of cap trapped first-strand cDNAs. As with previous versions of CAGE, we better define transcription start sites (TSS) than known models, identify novel regions of transcription and alternative promoters, and find two major classes of TSS signal, sharp peaks and broad regions. However, using this protocol, we observe reproducible evidence of regulation at the much finer level of individual TSS positions. The libraries are quantitative over 5 orders of magnitude and highly reproducible (Pearson's correlation coefficient of 0.987). We have also scaled down the sample requirement to 5 μg of total RNA for a standard HeliScopeCAGE library and 100 ng for a low-quantity version. When the same RNA was run as 5-μg and 100-ng versions, the 100 ng was still able to detect expression for ∼60% of the 13,468 loci detected by a 5-μg library using the same threshold, allowing comparative analysis of even rare cell populations. Testing the protocol for differential gene expression measurements on triplicate HeLa and THP-1 samples, we find that the log fold change compared to Illumina microarray measurements is highly correlated (0.871). In addition, HeliScopeCAGE finds differential expression for thousands more loci including those with probes on the array. Finally, although the majority of tags are 5′ associated, we also observe a low level of signal on exons that is useful for defining gene structures.