Technologies

A team of Stanford researchers has developed a highly sensitive, single molecule in situ hybridization system called Proximity Ligation- in situ hybridization (PLISH), which merges the specificity of proximity ligation, the sensitivity of probe tiling, and the high signal intensity of rolling circle amplification. PLISH enables efficient, high level multiplexing by iterative detect-image-erase cycles of four transcripts per round following a single hybridization step, with automated calculation of single cell expression profiles. Downstream data analysis can include clustering algorithms and t-SNE display, but unlike with tissue dissociative approaches for single cell profiling, spatial and morphological data is retained for each cell. Thus, PLISH enables spatial re-mapping of individual cells or cell classes back onto the tissue, allowing for inference of signaling centers, for instance, by localizing cells expressing ligands and cognate receptors. PLISH is highly versatile, performing well on archival FFPE (formalin fixed, paraffin embedded) or cryo-embedded tissue, and is compatible with conventional immunostaining for correlation of RNA and protein expression. PLISH is technically simple and inexpensive, employing conventional fluorescence microscopy and open source software, and can therefore be performed by any laboratory without requiring investment in new equipment. This technology can be used in combination with immunohistochemistry on primary tissue samples, including formalin-fixed and cryopreserved patient samples. Because PLISH is inexpensive and technically simple, it can be used by even non-expert biomedical researchers for in situ profiling to provide spatial context of newly-defined cell populations and reveal mechanistic aspects of human disease pathophysiology either alone or in conjunction with single-cell sequencing. In addition, it could potentially be adapted for diagnostics applications such as liquid biopsy or rare cell identification.