Gene expression profiling in single cells within tissue

Abstract

We developed a robust multiplex fluorescent in situ hybridization (FISH) technique in archival formalin-fixed, paraffin-embedded (FFPE) human tissue sections while preserving the microanatomical context. This identifies single-cell gene expression patterns by probing multiple, unique nascent RNA transcripts and yields predictive quantitative gene expression signatures.

Figure 1

Detection of nascent RNA (transcription sites) in paraffin-embedded tissue. (a) Detection of SMG1 (8 probes) transcription sites (arrows) in human PCa. Bar, 5 μm. (b) Detection of SMG1 (41 probes) nascent transcripts (arrows) in human PCa. Bar, 5 μm. (c) Colocalization of a gene and the respective chromosome. Prostate carcinoma exhibiting the colocalization of a DNA locus–specific probe for AR (androgen receptor, red spots) with the AR peT-FISH on a single section (green spots). Bar, 10 μm. (d) Multiplex detection of five genes in a prostate cancer sample (1982 archive), demonstrating restricted expression of four genes to the epithelial cells of the tumor annotated with the automated transcription site finder algorithm. The fifth gene in the barcoding scheme, EPB49, was only rarely detected and was not identified in this particular field. Bar, 10 μm.

Figure 2

Correlation of single-cell multigene expression profiles with diagnostic pathology. (a–c) Hematoxylin and eosin–stained images of focal PIN (a), PCa (b) and PCaMet (c), superimposed with the gene expression data from peT-FISH analyses of three genes (AR, JAG1 and FOLH1) obtained from the slide after destaining. Bars, 10 μm.

Figure 3

A combinatorial analysis of five genes provides a gene expression signature for each state. (a) The gene expression patterns of each diagnosis as a function of each gene (AMACR, AR, EPB49, JAG1 and FOLH1). (b) The gene expression patterns characteristic of each diagnosis (focal PIN, PCa, PCaMet and benign). Data represent the mean ± s.e.m.; n =20, 13, 14 and 12 for PCa, PIN, PCaMet and benign tissue, respectively.