Methods such as fluorescence in situ hybridization (FISH) allow gene expression to be observed at the tissue and cellular level; however, only a limited number of genes can be monitored in this manner, making transcriptome-wide studies impractical. George Church's group* is presenting the further development of their original approach called fluorescent in situ sequencing (FISSEQ) to incorporate a spatially structured sequencing library and an imaging method capable of resolving the amplicons (see Figure 1).
In fixed cells, RNA was reverse transcribed with tagged random hexamers to produce cDNA amplicons. Aminoallyl deoxyuridine 5-triphosphate (dUTP) was incorporated during reverse transcription and after the cDNA fragments were circularized before rolling circle amplification (RCA), an amine-reactive linker was used to cross-link the RCA amplicons containing aminoallyl dUTP. The team generated RNA sequencing libraries in different cell types, tissue sections, and whole-mount embryos for three-dimensional (3D) visualization that spanned multiple resolution scales (see Figure 1).
In a proof-of-concept experiment (see Figure 2) the authors sequenced primary fibroblasts in situ after simulating a response to injury, which yielded 156,762 reads, mapped to 8,102 annotated genes. When the 100 highest ranked genes were clustered, cells kept in fetal bovine serum medium were enriched for fibroblast-associated gene hits, while the rapidly dividing cells in epidermal growth factor medium were less fibroblast-like, reaffirming that the FISSEQ platform output reflects the change in transcription status as a function of the cellular environment and stress factors.
The authors further noted that FISSEQ appears to be sensitive to genes associated with cell type and function, and this in turn could be used for cell typing. It was also speculated that FISSEQ might allow for a combined transcriptome profiling and mutation detection in situ.
*Abstract from Science 2014, Vol. 343:1360–1363
Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample.
Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.