Universal qPCR Duplex Detection of miRNA and mRNA

MicroRNAs (miRNA) are a class of 21-23 ribonucleotides single strand RNAs and function as translation repressors by downregulating target mRNAs in a sequence-specific manner. Deregulation of miRNAs contributes to many diseases and can be used as robust biomarkers for the early diagnose, staging, prognosis and response to therapy of various human diseases. However, a method for accurate and sensitive detection of all miRNAs is lacking. A universal quantitative polymerase chain reaction (qPCR) duplex detection method was developed to simultaneously detect the expression levels of a specific miRNA and mRNA from two samples or two different RNAs in a PCR well with two distinctive universal fluorescent dual labeled hydrolysis probes. The expression levels of 96 miRNAs from the same sample quantitated by the fluorescein amidite (FAM) probe and the VIC probe are highly correlated (R2=0.98) indicating that this duplex detection method is highly reproducible and can be used to detect an identical miRNA in two different samples in a PCR well. A universal qPCR duplex detection of miRNAs and mRNAs were developed to detect differential expressions of miRNAs or mRNAs with high specificity and sensitivity in paired samples such as normal and diseased tissues. The well to well and plate to plate experimental variations can be greatly reduced, as the expression levels of an identical miRNA in two different samples are detected in the same well at the same time. The differential expression in two samples detected by this method is highly reproducible.