Sensimix sybr qpcr reagents

Total RNA was isolated using the miRNeasy Mini Kit (Qiagen; Valencia, CA, USA) and used to generate cDNA templates by reverse transcription of 1 μg RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Carlsbad, CA, USA) according to the manufacturer’s instructions. PCR reactions were performed in a total volume of 20 μL containing SensiMix SYBR qPCR reagents (Bioline; Taunton, MA, USA) using a Corbett Rotor-Gene 6000 real-time PCR system (Qiagen). PCR conditions consisted of an initial hold step at 95 °C for 10 min followed by 40 amplification cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. Sequences of the primer sets used were forward: 5′-CGGGAACAGGAACACACTAC-3′ and reverse: 5′-TTTGCCCTACACAAGGAAAG-3′ for ADRB1; forward: 5′-GAGCACAAAGCCCTCAAGAC-3′ and reverse: 5′-TGGAAGGCAATCCTGAAATC-3′ for ADRB2; forward: 5′-ACCATCCTCTTGCTCTCTGT-3′ and reverse: 5′-AGCCTGGAGAACACTAAGGT-3′ for ADRB3; and forward: 5′-GCTGCGGTAATCATGAGGATAAGA-3′ and reverse: 5′-TGAGCACAAGGCCTTCTAACCTTA-3′ for TATA-binding protein (TBP). The specificity of the amplified products was verified by melting curve analysis and agarose gel electrophoresis. qRT-PCR data were calculated as relative expression levels by normalization to TBP mRNA using the 2^−ΔΔCt method (Livak and Schmittgen 2001 (link)).