Abstract
Abstract The human genome encodes more than one thousand short (17-24 base) non-coding microRNA molecules which regulate diverse cellular functions. Several of these functions are also involved in tumorigenesis including cell growth, motility, and immune surveillance. Not surprisingly, numerous clinical studies suggest that microRNAs could serve as potent prognostic and/or predictive biomarkers for a variety of diseases including breast, colorectal, lung, and prostate cancers. However, due to their short sequence length, technical obstacles exist for detection of these potential biomarker targets particularly in FFPE tissue samples: (i) limited target sequence limits probe length and subsequent target specificity as well as (ii) detection sensitivity since short probes require significant amplification technologies to generate a visible signal; lastly, (iii) short microRNA targets may be less stable in tissue (increased ribonuclease sensitivity and/or limited crosslinking to cellular components). In the current study, new RNA detection technologies, which include hapten-labeled oligonucleotide probes and sensitive tyramide-hapten/Silver detection designed for detection of longer RNA targets, were adopted for automated detection of microRNA targets in formalin-fixed, paraffin-embedded (FFPE) tissue. DNP-labeled oligonucleotide probes, directed against four microRNA targets (miR-21, miR-200c, miR-25, and miR-200b) plus sequence-scrambled negative control were synthesized and combined with a modified VENTANA OptiView amplification kit and silver detection. The probes and new detection system catalyzed sensitive and specific detection of each microRNA target in MCF-7 xenograft FFPE tissues, which correlates well with microarray profiling data from MCF-7 cells by Fix et. al. (2010)*. In summary, this study demonstrates a robust and fully automated ISH assay for microRNA targets in FFPE tissue and enables potential microRNA biomarkers being analyzed within tissue context. * Fix et. al. Cancer Genomics & Proteomics (2010) 7:261-278. Citation Format: Zeyu (David) Jiang, Anne Pedata, Taylor Shingler, Lauren Behman, David Chafin, William Day. Automated microRNA detection in formalin-fixed, paraffin-embedded tissue samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 525. doi:10.1158/1538-7445.AM2014-525
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