Methods of miRNA detection and quantification by RT-qPCR. (a) A single miRNA is reverse transcribed using a gene-specific stem-loop primer. The resulting cDNA is then amplified using a cocktail of specific primers and a hydrolyzable probe. Procession of the Taq polymerase displaces, and hydrolyzes, the probe resulting in separation of the fluorophore and quencher. Accumulation of probe fluorescence signal is used to monitor the PCR reaction in real-time. (b) Alternatively all miRNAs are polyadenylated by poly(A) polymerase. Tailed miRNAs are then reverse transcribed using an oligo dT priming strategy. The resulting cDNA is amplified using specific primers (often containing LNA nucleotides to increase the primer Tm). The PCR reaction is monitored in real-time using a dye that fluoresces when bound to double-stranded DNA (e.g. SYBR green).