Tuesday, 6 December 2011: 13:15 - 13:30
Tulum (Cancún Center)
Jia-Wang Wang, PhD
,
Internal Medicine, University of South Florida, Tampa, FL
Kunyu Li, BS
,
Internal Medicine, University of South Florida, Tampa, FL
Richard F. Lockey, MD
,
Division of Allergy & Immunology, University of South Florida and James A. Haley Veterans' Hospital, Tampa, FL
Subhra Mohapatra, PhD
,
Nanomedicine Research Center and Divison of Translational Medicine, Depts of Molecular Medicine and Internal Medicine , University of South Florida and James V Haley Veterans' Hospital , Tampa, FL
Shyam Mohapatra, PhD
,
USF Nanomedicine Research Center, University of South Florida College of Medicine & VA Hospital, Tampa, FL
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., D.A. Ridzon, A.J. Broomer, Z. Zhou, D.H. Lee, J.T. Nguyen, M. Barbisin, N.L. Xu, 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 two 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 0.8ml 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.
