114 A Highly Sensitive and Specific Universal Mirna Profiling Method

BACKGROUND: miRNAs can be used as robust biomarkers for diagnosis, staging, prognosis and the response to therapy in various diseases. Although a wide spectrum of miRNA detection techniques have been developed, none can accurately and sensitively perform genome-wide high-throughput miRNA profiling (Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, et al 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33:e179). This problem stems from that miRNAs are only ∼22 bases, and multiple species of nucleic acids that contain the mature miRNA sequences are present in the total RNA samples that are usually used for miRNA detection. METHODS: A novel RT-qPCR miRNA assay (UQmiR, universally quantitating miRNA) was developed to overcome the difficulty. This assay requires only one RT reaction and one universal set of multiple hydrolysis probes to detect all miRNAs, using one universal RT primer, a common reverse primer, and individual miRNA-specific forward primers. A computer program (MSPPD, miRNA-specific primer and probe designer) was developed for the assay. RESULTS: The UQmiR has the advantages, but not the disadvantages, of the 2 mostly used miRNA assays. It has the specificity of hydrolysis probe assay and the universal detection of SYBR Green assay. This assay is more sensitive and specific than the commercially available hydrolysis probe assay and SYBR Green assay. Using this method, we have successfully detected 91 out of 96 miRNAs in 0.8 mL of plasma for each miRNA. CONCLUSIONS: This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.