Plasma miRNA Expression Profiling

Expression profiling of 188 miRNAs identified as the most consistent and reliable miRNAs in human plasma^{14 (link)} was performed using TaqMan-Low-Density-Array (TLDA) human custom arrays as instructed by the manufacturer (Applied Biosystem, CA, USA) with Applied Biosystems QuantStudio 7. Briefly, a fixed volume of 3 μL of miRNA solution was used for reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit and the TaqMan Custom RT pool (Applied Biosystems, CA, USA), which were customized for TLDAs. The reaction was performed under the following conditions: incubation for 30 min at 16 °C and 30 min at 42 °C, inactivation for 5 min at 85 °C and immediate cooling to 4 °C. The cDNA was stored at − 20 °C. For our custom selection, 3.125 μL of the reverse transcription reaction was used for preamplification using TaqMan PreAmp Master Mix (2X) and TaqMan PreAmp pool (Applied Biosystems, CA, USA). The reaction conditions were as follows: 10 min at 95 °C, 2 min at 55 °C, 2 min at 72 °C, 12 cycles of 15 s at 95 °C and 4 min at 60 °C, inactivation for 10 min at 99.9 °C and cooling to 4 °C. The preamplification reaction was then diluted with water (1:3), and 4.5 μL was used for TLDA. Reverse transcription and preamplification reactions were carried out with an Applied Biosystems Verity Thermal Cycler. Data were normalized by the mean-centre normalization method.