Mycycler pcr thermal cycler system

Total cellular RNA was extracted using Trizol (Invitrogen). The ratio of the absorbance at 260 nm and 280 nm (A260/A280) was used to assess RNA purity. Reverse transcription was performed with 0.2 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Beijing TransGen Biotech Co. Ltd., China). PCR was performed using a MyCycler™ Thermal Cycler PCR system (Bio-Rad, Hercules, CA, USA) using: AQP7: forward 5′-GAA CAG TGA GAA AAA GAC CG-3′ and reverse 5′-CCA GAC AAT CCA GAG TTC AT-3′; 18S ribosomal RNA (18S rRNA): forward 5′-TTG GTG GAG CGA TTT GTC TG-3′ and reverse 5′-AAT GGG GTT CAA CGG GTT AC-3′). All reactions involved initial denaturation at 95 °C for 5 min followed by 36 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s. PCR products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and analyzed using the Alpha Innotech software (Bio-Rad). A kinetic study was performed using different numbers of cycles in order to determine the optimal number of cycles for mRNA quantification during the exponential phase. We observed that 36 cycles provided the best results. AQP7 expression was normalized to the 18S rRNA expression level, and was quantified using a standard curve. There was no significant difference in baseline 18S rRNA levels between control and apelin-13- or palmitate-treated adipocytes.