MiR profiling of the detection cohort using MicroRNA Arrays

Comparative RT-PCR-based TaqMan low-density miR arrays A and B were applied for profiling of 3 μl samples containing 1000 ng of RNA. Reverse transcription reaction of the extracted RNA was performed using Megaplex RT Primer A and B and TaqMan™ microRNA RT Kit (Thermo Fisher Scientific, Darmstadt, Germany) as previously described⁴⁹. 1 µg of cDNA was loaded on TaqMan™ Human MicroRNA Arrays Cards V2.0 (Set A and B) for miR profiling of 754 miRs with snoU6 as endogenous control (Thermo Fisher Scientific, Darmstadt, Germany). Quantification was performed by TaqMan™ ABI PRISM 7900HT Sequence Detection System (Thermo Fisher Scientific, Darmstadt, Germany). A total of 100 μl master mix containing 100 ng cDNA was loaded into each of the eight ports. The distribution into 48 reaction cavities per port was carried out by two short centrifugation steps (1 min 1200 rpm in a swinging bucket rotor, Heraeus-Multifuge-3S, Langensebold, Germany). Cross contamination was avoided by individual sealing of the 384 reaction cavities. Cycling conditions were as followed: Activation step at 92 °C for 10 min, followed by 40 PCR cycles at 95 °C for 1 sec and 60 °C for 20 sec.