As regulators of a large number of human genes microRNAs are involved in various cellular processes, such as cell growth, proliferation, apoptosis and angiogenesis. Thus, the deregulation of microRNA expression has been linked to several diseases, including cancer. Intratumoural heterogeneity and increasing evidence that minor subpopulations of the tumour or the tumour microenvironment can be clinically significant, point out the need of cell-based tools for microRNA analysis. The up-regulation of miR-10b expression in a small subset of tumour cells for example has been implicated in the occurrence of EMT and triggering of tumour invasion in breast cancer. Therefore, determining the cell specific expression and localization of microRNAs, such as miR-10b will contribute to a better understanding of mechanisms of action of miR-10b and its role cancer spread.
Using breast and colon cancer cell line cells we established a novel one-day protocol for combined in situ hybridization (ISH) and immunohistochemistry (IHC), that enables the analysis of the expression and localization of microRNAs in individual cells. Target miRNAs were visualized using double-DIG labelled probes and FITC-labelled anti-DIG antibodies. Stringent hybridization and washing conditions were optimised to allow specific and efficient probe binding, but to avoid damaging important antigens for following IHC detection. Using this protocol we were able to analyse the expression of microRNAs, such as miR-10b in cell subpopulations of fresh frozen tumour sections and in tumour cells detected in peripheral blood samples.
In summary, our protocol allows multivariate microRNA expression analysis of individual cells and cell subpopulations, which can be immunohistochemically detected in a sample in a one-day process.