We determined the amounts of the individual miRNAs by real-time quantitative RT-PCR with a TaqMan MicroRNA Assay (Thermo Fisher Scientific) and the following miRNA-specific stem-loop primers: hsa-miR-148b (000471), hsa-miR-326 (000542), hsa-let7b (000378), and RNU6B (001093). Subsequently, quantitative real-time PCR was performed with an ABI Prism 7000 sequence detection system (Thermo Fisher Scientific). The reaction was initiated by incubation at 95°C for 2 minutes, followed by 50 cycles of 95°C for 15 seconds and then 60°C for 1 minute. All reactions were run in duplicate. Mean (Ct) values for all miRNAs were quantified with sequence detection system software (SDS version 1.02; Thermo Fisher Scientific). All miRNA expression was normalized to RNU6B expression, yielding a −ΔCt value, as reported elsewhere.20 (link) The −ΔΔCt value was then calculated by subtracting the −ΔCt value of the normal sample from the respective −ΔCt values of patient samples. Expression of all miRNAs was normalized by using the 2−ΔΔCt method.