Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ
Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ
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DOI：
10.1101/gr.234807.118
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发表时间：
2019-11-01
期刊：
影响因子：
7
通讯作者：
West, Robert B.
中科院分区：
文献类型：
--
作者：
Foley, Joseph W.;Zhu, Chunfang;West, Robert B.
RNA测序(RNA-seq)是一种灵敏准确的基因表达定量方法。小样本或其RNA被降解的样本，如福尔马林固定的石蜡包埋(FFPE)组织，对于使用非专门的RNA-SEQ方案进行研究仍然具有挑战性。在这里，我们提出了一种新的方法，Smart-3SEQ，它即使只有少量的总RNA也能准确地定量转录丰度，并有效地表征通过激光捕获显微切割(LCM)从FFPE组织中提取的小样本。我们还从FFPE单细胞中获得了不同的生物学特征，这是以前的RNA-SEQ方案无法研究的，我们使用这些数据来识别可能与肿瘤微环境相关的新的巨噬细胞表型。我们建议Smart-3SEQ作为一种高成本效益的方法，使大型基因表达谱实验不受样本大小和组织可用性的限制。特别是，Smart-3SEQ与FFPE组织的兼容性解锁了大量存档的临床样本；与LCM相结合，它允许对按原位环境分离的小细胞群和单细胞进行前所未有的研究。
RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.