Quantitative PCR Analysis of Gene Expression

Blood was collected and prepared as described using the PAXgene Blood RNA system (Qiagen,Valencia, CA, USA)[28] (link). Samples with RNA integrity values > 7.0 and ratio of absorbances at 260/280 nm between 1.7 and 2.4 were used in the current study. Primer Express software (Life Technologies, Carlsbad, CA, USA) was used to design the primers. The High Capacity RNA transcription kit (Life Technologies, Carlsbad, CA, USA) was used to reverse transcribe 1 µg of total RNA according to the manufacturer's protocol. The DNA engine Opticon 2 Analyzer (Bio-Rad Life Sciences, Hercules, CA, USA) was used for the qPCR reactions. Each 25 µl reaction contained Power SYBR (Life Technologies, Carlsbad, CA, USA) and primers at a concentration of 5 µM. Primer sequences used in qPCR assays are as follows: GAPDH; forward: 5′- CAACGGATTTGGTCGTATTGG-3′; reverse: 5′- TGATGGCAACAATATCCACTTTACC-3′, SOD2; forward: 5′- GTTCAATGGTGGTGGTCATATCA-3′; reverse: 5′- GCAACTCCCCTTTGGGTTCT-3′. Amplification conditions and detailed description of qPCR experiments is described in [15] (link).