Sensitive and Specific Universal RT-qPCR Profiling of miRNAs

Abstract

RATIONALE: MicroRNAs (miRNAs) bind to target mRNAs and regulate their expression in a dose-dependent manner. The amount of specific miRNA in the cell is critical for the regulation process. Their expression pattern can be used as signatures of the developmental stage of cells and tissues, and as a biomarker for diagnosis and prognosis of disease, and miRNAs may be useful as therapeutic agents. Accurate quantitation of miRNAs is essential to their study. However, a highly specific and sensitive but simple method for quantitating miRNAs is still sought. METHODS: The method combines a universal hydrolysis probe with a universal reverse trasncription primer (UPR), which requires only one RT reaction and one hydrolysis probe for detection of all miRNAs. A computer program has been developed to design the probes and primers for the RT-qPCR assay. RESULTS: The method can detect and quantify miRNAs in as little as 1 pg of total RNA, and genomic DNA and mRNAs present in total RNA produce negligible or undetectable signals. Simple extraction methods such as Trizol (Invitrogen, CA, USA) without DNase treatment and small RNA fractionation is sufficient to produce total RNA for this assay. CONCLUSION: This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.