RNA was purified from T-Per or M-Per lysates using the miReasy kit (Life Technologies, Grand Island, NY, USA) or Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. Quality of RNA was determined in a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (all RINs ≥8.5). cDNA was produced from 500ng RNA using the NCODE Vilo cDNA systhesis kit (microRNAs) or SuperScript III First-Strand Synthesis kit (mRNAs) following the manufacturer’s instructions (Life Technologies).
For qPCR, cDNA (1/10 dilution) was amplified on a MyIQ thermocycler (Biorad) using the SensiMix SYBR & Fluorescein kit (Bioline, Taunton, MA, USA) on the following conditions. microRNAs: 95°C 10min; 40x (95°C 15 sec, 60°C 30 sec); dissociation curve 55°C-95°C with 0.5°C increments every 10 sec. mRNAs: 95°C 10min; 35x (95°C 30 sec, 60°C 30 sec, 72°C 30 sec); dissociation curve 55°C-95°C with 0.5°C increments every 10 sec. Data was corrected by efficiency [38 (link)] using the LinRegPCR software [39 (link)] and normalized to RNU6 (microRNAs) or GAPDH (mRNAs) levels. Primer sequences were selected using the Primer3 software [40 ] or from the Invitrogen website. A complete list of the primer sequences used in the present study is shown intable 1 .
For qPCR, cDNA (1/10 dilution) was amplified on a MyIQ thermocycler (Biorad) using the SensiMix SYBR & Fluorescein kit (Bioline, Taunton, MA, USA) on the following conditions. microRNAs: 95°C 10min; 40x (95°C 15 sec, 60°C 30 sec); dissociation curve 55°C-95°C with 0.5°C increments every 10 sec. mRNAs: 95°C 10min; 35x (95°C 30 sec, 60°C 30 sec, 72°C 30 sec); dissociation curve 55°C-95°C with 0.5°C increments every 10 sec. Data was corrected by efficiency [38 (link)] using the LinRegPCR software [39 (link)] and normalized to RNU6 (microRNAs) or GAPDH (mRNAs) levels. Primer sequences were selected using the Primer3 software [40 ] or from the Invitrogen website. A complete list of the primer sequences used in the present study is shown in
