Abstract 2021: MicroRNA affinity assay

Abstract

Abstract Introduction: MicroRNAs (miRNAs) are small noncoding RNAs functioning in gene regulation. In animal cells, miRNAs bind to target sites in the 3’ UTR of mRNAs, causing posttranscriptional repression or degradation of mRNAs. The identification of specific miRNAs to target a 3′UTR of a given gene is essential for studies in gene regulation. However, the sequence software programs conventionally used to predict binding of individual microRNAs to a given mRNA transcript are notoriously unreliable, and specific microRNA-mRNA binding is very difficult to directly affirm using currently available techniques. We have developed a microRNA affinity assay that addresses this need. Methods: We chose a candidate gene that had been extensively studied experimentally for microRNA regulation; it is known that miR-200c and miR-200b can bind to 3′UTR RNA of TCF8 [Hurteau 2007]. Here, a 500bp fragment of the 3′UTR RNA of TCF8 was generated by IVT, and used as a bait sequence to identify miRNAs that bind a mixture of known miRNA composition, and alternately a naturally produced cellular miRNA pool. After separation, miRNA qPCR was used to check recovery rate of miRNA, and cloning and sequencing was used to identify miRNAs. Results: Specific miRNAs from a synthetic mixture miRNAs, miRNA qPCR results displayed excellent recovery rates (151.57% with pipetting error, for miR-200c), 88.88% for the closely related miR-200b, 0.31% for the sequence-unrelated miR-9, 0.67% (miR-10a), 0.1% (miR-20a), 8.78% (miR-34a with some 3′UTR homology), 0.11% (miR-100), 0.16% (miR-140-5p), 0.98% (miR-144), 0.33% (miR-802) and 1.58% (miR-1234). After cloning the captured miRNAs, there were 10 sequences from miR-200c, 7 sequences from miR-200b, and one other sequence in 18 sequenced clones. When separating specific miRNAs from naturally produced NHBE cell miRNA pool, miRNA qPCR results show that their recovery rates are 88.88% (miR-200c), 80% (miR-200b), 0.07% (miR-20a), less than 0.006% (miR-34a) and 0.01% (miR-100). Conclusions: For both simple known and complex unknown microRNA pools, this pull-down strategy is effective in determining microRNA-mRNA binding. In future work, we will use other 3′UTR RNA sequences of candidate genes as bait sequence, for microRNA-related discovery and confirmation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2021.