Abstract 4055: Real-time quantitation of primary transcripts of microRNA genes

Regulation of mature microRNA (miRNA) expression has been shown during the course of biogenesis under different contexts: from transcription, processing of pri-miRNA to pre-miRNA by Drosha, to processing of pre-miRNA to mature miRNA by Dicer. A mature miRNA can be processed from two or more stem-loop precursor loci, which are denoted with numeric suffixes such as −1, −2, etc. As of the 14 th Release of the miRBase, there are 154 genes among the 750 miRNA genes that belong to this category. To interrogate regulation of miRNA expression at the transcriptional level for those that can be processed from multiple genomic locations, it requires assays to specifically quantitate the precise locus of interest. We have developed a pipeline to design and select real-time RT-PCR assays to quantitate primary transcripts of miRNA genes. These assays are designed against genomic DNA sequence so the genomic DNA needs to be removed by prior enzyme treatment. We showed that the TaqMan® Pri-miRNA Assays have minimally detectable or no background using either cell lysates or commercial tissue total RNA. Excellent linearity was seen using cDNA synthesized from total RNA, or using 1ng/ul or greater of genomic DNA. The specificity of the assays to distinguish loci that are processed into identical mature miRNA was demonstrated using subcloned plasmids, as well as using breast cancer cell lines that showed concordant expression patterns among such loci with published data in human breast cancer. The TaqMan® Pri-miRNA Assays will be a useful tool to identify the precise loci that contribute to the mature miRNA expression of interest at the transcription level, so as to further investigate the regulatory mechanisms for the differential expression. 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 4055.