Mycycler gradient

Total cellular RNA, including microRNA and other small RNA molecules, was isolated using the miRNeasy RNA kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The quality and quantity of the RNA eluted were measured using a spectrophotometer (Nanodrop 1000 V 3.6, Thermo Fisher Scientific, Waltham, MA, USA). The ratio A₂₆₀/A₂₈₀ was used to assess RNA purity, considering samples with a ratio between 1.9–2.0 as pure RNA. For cDNA synthesis, the RT² First Strand kit (Qiagen, Germantown, MD, USA) was employed, using 0.5 µg of total RNA as a template. First, samples were incubated in a thermocycler (MyCycler Gradient, Bio-Rad, Hercules, CA, USA) for 5 min at 42 °C to eliminate genomic DNA, followed by two incubations of 15 min at 42 °C and 95 °C for 5 min in a final volume of 20 µL. Afterward, 91 μL of RNA-free water was added to each reaction and the samples were kept on ice until proceeding with the real-time PCR (RT-PCR) protocol.

In order to perform microRNA expression analysis, RNA samples were polyadenylated by poly(A) polymerase and subsequently converted into cDNA by a reverse transcription reaction using an oligo-dT primer with universal tag sequence on the 5′ end (miScript II RT Kit Qiagen, Germantown, MD, USA). For cDNA synthesis, 250 ng of total RNA were mixed with the different components according to the manufacturer’s instructions and were incubated in a thermocycler (MyCycler Gradient, Bio-Rad, Hercules, CA, USA) following a two-step reaction: 60 min at 37 °C and 5 min at 95 °C to inactivate reverse transcriptase activity. Finally, each sample was diluted in 200 µL of RNase-free water and stored at −20 °C until use.