RNA was purified from T-Per or M-Per lysate using the miRNeasy kit (Life Technologies) or Direct-zol RNA MiniPrep kit (Zymo Research) following the manufacturer's instructions. Quality of RNA was determined in a 2100 Bioanalyzer (Agilent Technologies) (all RINs ≥8.5). cDNA was produced from 500 ng RNA using the NCode VILO cDNA synthesis kit (miRNAs) or SuperScript III First-Strand Synthesis kit (mRNAs) following the manufacturer's instructions (Life Technologies).
For qPCR, cDNA (1/10 dilution) was amplified on an MyiQ thermocycler (Bio–Rad Laboratories) using the SensiMix SYBR & Fluorescein kit (Bioline) in the following conditions: miRNAs: 95°C, 10 min; 40× (95°C, 15s; 60°C, 30 s); dissociation curve 55–95°C with 0.5°C increments every 10 s. mRNAs: 95°C, 10 min; 35× (95°C, 30 s; 60°C, 30 s; 72°C, 30 s); dissociation curve 55–95°C with 0.5°C increments every 10 s. Data were corrected efficiently by using the LinRegPCR software and normalized to RNU6 (miRNAs) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mRNAs) levels.
For qPCR, cDNA (1/10 dilution) was amplified on an MyiQ thermocycler (Bio–Rad Laboratories) using the SensiMix SYBR & Fluorescein kit (Bioline) in the following conditions: miRNAs: 95°C, 10 min; 40× (95°C, 15s; 60°C, 30 s); dissociation curve 55–95°C with 0.5°C increments every 10 s. mRNAs: 95°C, 10 min; 35× (95°C, 30 s; 60°C, 30 s; 72°C, 30 s); dissociation curve 55–95°C with 0.5°C increments every 10 s. Data were corrected efficiently by using the LinRegPCR software and normalized to RNU6 (miRNAs) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mRNAs) levels.