Sybr green real time pcr kit

Total RNA was extracted from the tissue samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, US). Subsequently, complementary DNA was synthesized using a reverse transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers’ instructions. The relative expression levels of mRNA were determined using a SYBR Green real-time PCR kit (Agilent Technologies, Inc., Santa Clara, CA, USA) and normalized to GAPDH. RT-PCR was performed using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the following gene-specific primers: GAPDH sense: 5′-TGCCATCAACGACCCCTTCA-3′; GAPDH anti-sense: 5′-TGACCTTGCCCACAGCCTTG-3′; Sirt1 Sense: 5′-GAGGCAGTGCAGCATGTAGT-3′, Anti-sense 5′-GATGATTCCCTCGGTCAGAA-3′. All primers were designed using the National Center for Biotechnology Information Primer-BLAST tool. PCR was performed under the following conditions: Denaturation at 50°C for 2 min, followed by 35 cycles of 94°C for 15 s and 56°C for 45 s. Gene expression was normalized to internal controls and fold changes were calculated using relative quantification (2^−ΔΔCt).