Expression Microdissection for use in qPCR based analysis of miRNA in a single cell type

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.