The noncoding RNA designated as microRNA (miRNA) is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH) procedures to detect miR-146a with (a) different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA) incorporated DNA oligonucleotides; (b) different reporters for the probes: biotin versus digoxigenin (DIG); (c) different visualization: traditional versus tyramide signal amplification (TSA) system; (d) different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC) and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value. 1. Introduction MicroRNA refers to the category of single-stranded small noncoding RNAs that are approximately 22 nucleotides in length. More than 1500 human microRNA have been identified and registered via various approaches including high throughput screenings (http://www.mirbase.org/). As more than 30% of human of mRNAs are regulated by microRNAs, the functional impact of microRNA in physiology and pathology has yet to be fully elucidated [1]. Generally speaking, microRNA imposes its regulatory role by sequence-specific but incomplete complementary binding to its target mRNA sequences, which are usually located at the 3′ untranslated region [1, 2]. This binding may mediate the degradation of target mRNA or the inhibition of protein translation efficiency of target mRNA. However, due to the loose stringency of this kind of targeting, the exact mechanism of specific microRNA function remains undefined and is an actively addressed research topic. The function and target mRNAs of individual microRNAs cannot be reliably predicted via current bioinformatic approaches, thereby warranting continued experimental interrogation. The investigation of microRNA expression and
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