Strata gene mx3000p real time pcr system

Total RNA was isolated using TRIzol® Reagent (Invitrogen, Cat#15596-026) according to the manufacturer’s recommendations. RNA concentrations were quantified and reverse-transcribed using ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Cat#11146-016). Gene expressions were detected using GoTaq qPCR Master Mix (Promega, Cat#A6001) in Strata gene MX3000P Real-Time PCR system (Genetimes, China). Relative gene expression levels were calculated by △△Ct and compared with GAPDH as internal control.

Total RNA was isolated using TRIzol^® Reagent (Invitrogen, Cat# 15596-026) according to the manufacturer's recommendations. RNA concentrations were quantified and reverse-transcribed using ThermoScript^TM RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Cat# 11146-016). Gene expressions were detected using GoTaq qPCR Master Mix (Promega, Cat# A6001) in Strata gene MX3000P Real-Time PCR system (Genetimes, China). Relative gene expression levels were calculated by ΔΔCt and compared with GAPDH as internal control.
Primers were designed using Primer 5.0 software: PPARγ (NM_001145366; forward: 5′-GTCACACTCTGACAGGAGCC-3′; reverse: 5′-CAC CGCTTCTTTCAAATCTTGT-3′), GAPDH (NM_017008; forward: 5′-AAGGGCTCATGACCACAGTC-3′; reverse: 5′-CAGGGATGATGTTCTGGGCA-3′), OBRb (AF287268; forward: 5′-TCCAGGTGAGGAGCAAGAG-3′; reverse: 5′-TTCAGCGTAGCGGTGATG-3′), and STAT3 (NM_012747; forward: 5′-TATCTTGGCCCTTTGGAATG-3′; reverse: 5′-GTTGTAGGACCATAGGGGTG-3′). Each reaction mixture (25 μL total volume) contained 12.5 μL Maxima SYBR Green Master mix (2X), 0.3 μM of each primer, nuclease-free water, and 20 ng template DNA. Amplification of each gene was performed in duplicate runs and PCR conditions were 95°C for 10 min, followed by 40 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C.