Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ

Genome Res. 2019 Nov;29(11):1816-1825. doi: 10.1101/gr.234807.118. Epub 2019 Sep 13.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Profiling / methods*
  • Humans
  • Laser Capture Microdissection / methods*
  • Macrophages / metabolism
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Tumor Microenvironment