Icycler abi viiatm7

Total RNA was isolated from HBE cells using the RNAiso Plus kit (Takara Biotechnology Co. Ltd., Dalian, China), according to the manufacturer's instructions. cDNA was synthesized from the RNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co. Ltd., Dalian, China). Real-time qPCR was performed using SYBR®Premix Ex Taq^TM II (Tli RNaseH Plus) (Takara Biotechnology Co. Ltd., Dalian, China) on an iCycler (ABI ViiATM7; Applied Biosystems, Carlsbad, CA, USA). The thermocycler parameters were set as follows: step one, activation of the HotStartTaq DNA polymerase (Takara Biotechnology Co. Ltd.) at 95°C for 30 sec; step two, PCR was performed for 40 cycles with denaturation at 95°C for 5 sec and annealing at 60°C for 34 sec; step three, fixed parameters set by the ABI 7500 Fast Real-time PCR system (Applied Biosystems)-associated SDS software version 2.3 (Applied Biosystems). Data were quantitated with 2^−ΔΔCt.
For each gene, an amplification curve was generated to evaluate the amplification efficiency. The sequences of the forward and reverse primers were: MFN2 forward 5'-CAGGTGTAAGGGACGATTGG-3' and reverse 5'-CAAATGGGATGAAGCACTGA-3'; GAPDH forward 5'-CAAATGGGATGAAGCACTGA-3' and reverse 5'-CGTCAAAGGTGGAGGAGTG-3'.

Total RNA was isolated from HBEpCs using TRIzol^® (Invitrogen Life Technologies, Beijing, China), according to the manufacturer’s instructions. cDNA was synthesized from the RNA using the Superscript II Reverse Transcription system (Invitrogen Life Technologies). qPCR was performed using a SYBR Green PCR kit (Takara Biotechnology Co. Ltd., Dalian, China) using an iCycler (ABI ViiATM7; Applied Biosystems, Carlsbad, CA, USA). The thermocycler parameters were set as follows: Step one, activation of the HotStartTaq DNA polymerase (Takara Biotechnology Co. Ltd.) at 95°C/30 sec; step two, PCR was performed for 40 cycles, denaturation at 95°C/5 sec, annealing at 60°C/34 sec; step three, fixed parameters set by the ABI ViiA 7 Fast Real-time PCR system (Applied Biosystems). GAPDH was used as an internal standard. The primer sequences were designed using the NCBI-Primer Basic Local Alignment Search Tool (National Institutes of Health, Bethesda, MD, USA) online tool and synthesized by Sangon Biotech, Shanghai, Co., Ltd. (Shanghai, China). The oligonucleotides used were as follows: SIRT1 forward, 5′-ATTCCAGCCATCTCTCTGTCAC-3′, and reverse, 5′-GTCTTGTATCTGTGCGACCTTG-3′; ORP150 forward, 5′-CAGAGGGAGAGAAGAAGCAGAA-3′, and reverse 5′-CAAGACCTGGACGGACTGAA-3′; GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′, and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′.