Iscript reverse transcription mastermix

Total RNA was extracted from PFC tissue using PureZOL reagent (Bio-Rad, USA) according to the manufacturer's instructions. Reverse transcription was done using iScript Reverse Transcription Mastermix (Bio-Rad, USA). Quantitative RT-PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA). mRNA specific primers for Bax, Bcl-2, BDNF, anti-GABA A receptor alpha 5/GABRA5, and β-actin as a housekeeping gene were used (Supplementary file (available here)). Quantitative RT-PCR reactions were done in Applied Biosystems 7500 (Applied Biosystems, USA), and after data analysis, the relative gene expression was calculated according to Livak and Schmittgen [39 (link)].

Total RNA was extracted from the liver, renal, and testicular tissues using a PureZOL reagent (Bio-Rad, USA) according to the manufacturer's instructions. Reverse transcription was done using iScript Reverse Transcription Mastermix (Bio-Rad, USA). Quantitative RT-PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA). mRNA-specific primers for Bax, Bcl-2, and β-actin as a housekeeping gene were used. Quantitative RT-PCR reactions were done in the Applied Biosystems 7500 (Applied Biosystems, USA), and after data analysis, relative gene expression was calculated according to Livak and Schmittgen [33 (link)].

Bone specimens were excised and snap frozen in liquid nitrogen prior to homogenization. Total RNA from the bone specimens was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. For reverse transcription, iScript Re-verse Transcription Mastermix (Bio-Rad, Hercules, CA, USA) was used. Real-time PCR was carried out using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer protocol, and mRNA-specific primers (Table 1) for OPG, RANKL and β-actin as a housekeeping gene (Invitrogen, Waltham, MA, USA). All samples underwent the same RT-PCR protocol: activation at 95 °C for 30 s followed by 40 cycles including denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s. Quantitative RT-PCR reactions were done in the Applied Biosystems 7500 (Applied Biosystems, Waltham, MA, USA). Quantitative RT-PCR reactions were performed in duplicate, and the mean values were further analyzed. After data analysis, relative gene expression was calculated according to Livak and Schmittgen [43 (link)].