Research Solutions: Single-Cell Analysis

Researchers investigating highly heterogeneous tumors are familiar with discrepancies between single-cell and average cell measurements. 'Grind-and-bind' methods like qRT-PCR yield data averaged from a population of cells that have been homogenized and are not representative of the actual transcriptional status of any individual cell. Even when used with an enrichment method like laser capture microdissection to specifically capture and enrich the specimen for cells of interest prior to analysis, the morphological context of the surrounding tissue is lost. The loss of morphological context, which provides valuable and often critical information for disease diagnosis by standard histopathological examination, can lead to clinical misinterpretation.

In order to fully understand the cellular specificity and complexity of tissue microenvironments in clinically relevant histopathological FFPE specimens, it is necessary to measure molecular signatures with single-cell resolution and in situ. RNAscope® in situ hybridization assays are ideal to study gene expression profiles within and between single cells in complex biological samples and FFPE tissue sections. One of the key advantages of this method is that gene expression of multiple genes can be analyzed in single cells in situ and quantified using SpotStudio™ Software, providing quantitative molecular characterization of a single cell within the complex cellular and morphological context.

Single-cell analysis is crucial for elucidating cellular diversity and heterogeneity, not only in tissue specimens but also in “liquid biopsy” specimens, such as circulating tumor cells (CTCs) and peripheral mononuclear blood specimens (PBMCs). Two applications of RNAscope® Technology have been demonstrated for CTC analysis (CTCscope™) and for PBMC expression analysis using flow cytometry (RNAflow™). These novel applications of multiplex in situ hybridization for determination of expression in rare cells and using flow cytometry, in combination with quantitative analysis, will usher in the next generation of single-cell analysis.

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