Circulating miRNA Profiling in Non-Muscle Invasive Bladder Cancer

Global miRNA profiling was performed on serum samples from ten NMIBC patients and ten sex- and age-matched healthy subjects using TaqMan^® Array Human MicroRNA Card Set v3.0 (Applied Biosystems, Foster City, CA, USA), which contains probes for 754 human miRNAs. Briefly, total circulating RNA was reverse-transcribed using TaqMan^® MicroRNA Reverse Transcription Kit and the Megaplex™ RT Primers followed by a pre-amplification reaction using TaqMan^® PreAmp Master Mix and Megaplex PreAmp Primers. Preamplified target cDNAs were then mixed with TaqMan^® Universal PCR Master Mix and loaded onto the TaqMan^® MicroRNA Array. Quantitative real-time PCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Expression threshold for each miRNA detector was automatically determined. After initial screening, a total of 166 miRNAs could be detected. Among these, we selected 89 miRNAs that are related to cancer pathways or reported in bladder cancer based on literature [57 (link)–62 (link)] for further testing in discovery and validation sets (Supplementary Table 4). MiRNAs with a Ct value < 35 and a missing rate < 25% in both study groups were considered as stably detectable candidates for further analysis.