miRNA and mRNA Reverse Transcription Protocol

Reverse transcription for miRNA was done with a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Two nanograms/microliter of RNA was added to 0.15 μL of 100 mM dNTPs (with dTTP), 1.00 μL of MultiScribe™ Reverse Transcriptase (50 U/μL) (ThermoFisher Scientific K. K., Kanagawa, Japan), 1.50 μL of 10 × Reverse Transcription Buffer, 0.19 μL of RNase inhibitor (20 U/μL), and 3.0 μL of RT primer; distilled water (dH₂O) was added to make 10 μL. Reverse transcription was performed using a Master Cycler Gradient (Eppendorf) at 16 °C for 30 s, 42 °C for 30 min, and 85 °C for 5 s and the product was stored at 4 °C. Reverse transcription for mRNA was performed according to the previously described method [10 (link)].