Quantitative RT-qPCR for microRNA Profiling

Quantitative real-time RT–PCR analysis was done with an ABI Prism 7500 Sequence Detection System using the miRCURY LNA™ Universal RT cDNA Synthesis Kit (Exiqon). The cDNA was diluted 50X and assayed in 10 µl PCR reactions according to the protocol for the miRCURY LNA™ Universal RT microRNA PCR System (Exiqon A/S); each microRNA was assayed twice by qPCR on the plasma Focus microRNA PCR panel. A no-template control (NTC) of water was purified with the samples and profiled like the samples. Analysis of the data was performed using the relative miRNA expression according to the comparative Ct (ΔΔCt) method using negative metastatic samples as reference. We used the geNorm (37 (link)) or the Normfinder algorithm (38 (link)) to select the best combination of two reference genes. Data from multiples plates were normalized using UniSp3 spike-in (Exiqon) as interplate calibrators.