Schematic representation of stem-loop microRNA quantitative RT-PCR.

The two main steps are reverse transcription and real-time PCR. In the first step, mature miRNA is extended and reverse transcribed by a sequence specific stem-loop primer. In the second step, the reverse transcribed miRNA is quantified by a fluorescently labeled hybridization probe using the strand replacement reaction. According to the previous protocol, all targets (e.g. endogenous control and target) should be reverse transcribed separately. In the dual-labeled probe based detection systems Q stands for quencher, F for fluorophore. Red exclamation marks indicate crucial points of the procedure that are discussed in this paper.