High profile 96 well pcr plate

Tissue samples of BAT of the mice were homogenized, phase separation was performed, and total RNA was isolated using the Trizol (Ambion, life technologies, Carlsbad, USA) method according to the manufacturers' protocols. RNA was further purified with the RNeasy Mini Kit (Kit 74104, Qiagen, Hilden, Germany). RNA quantification and purity was evaluated with Nanodrop (Thermo Scientific, Waltham, USA). Five micrograms of RNA were reverse transcribed into cDNA using MultiScribe reverse transcriptase and random primers (Kit 4368814, Applied Biosystems, Vilnius, Lithuania). The PCR reaction was performed in a mixture that contained appropriate forward and reverse primers and the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, USA) as the indicator. Real-time PCR amplification and data analysis were performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, USA). Genes were assayed in triplicate for each sample in a High-Profile 96-Well PCR Plate (Bio-Rad Laboratories, Hercules, USA). The data were analyzed using the ΔCt-method and normalized to internal housekeeping gene control of cyclophilin A mRNA to determine relative gene expression levels (mean expression level in the respective control group was set to 1.0). All primer sequences used are shown in ▶Table 2S.