2.6. PCR Amplification

Total RNA was extracted using TriZol (Invitrogen) according to the manufacturer's instructions. Reverse transcription (RT) reactions were carried out with Omniscript RT kit (Qiagen, Mississauga, ON, Canada) using random hexamers (Invitrogen). PCR amplifications for semi-quantitative analysis were conducted with Taq PCR core kit (Qiagen) using specific primer sets for murine ApoD (F: 5′-CCACCGGCACCCTACTGGATC-3′, R: 5′-CGGGCAGTTCGCTTGATCTGT-3′) and human ApoD (F: 5′-TTAACCTCACAGAGCCTGCC-3′, R: 5′-GAGTCCACTGTTTCTGGAGGG-3′); Gapdh (F: 5′-AGAACATCATCCCTGCATCC-3′, R: 5′-AGTTGCTGTTGACGTCGC-3′) was used as a reference gene for normalization. Amplifications were carried out for 40 cycles of 1 min at 94 °C, 30 s at 58 °C and 1 min at 72 °C. PCR products were resolved in 2% agarose gel and visualized under UV with Redsafe (FroggaBio, North York, ON, Canada) staining. Real-time PCR analysis for mouse ApoD, Opg, Rankl and Sod1 was performed using the iCycler IQ detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green I (Bio-Rad) as a double-strand DNA-specific binding dye, using β-microglobulin (B2m) as a reference gene (F: 5′-TACTCAGCCACCCACCGGCCG-3′, R: 5′-GCTCGGCCATACTGGCATGCT-3′) and the following sets of specific primers: Opg (F: 5′-GTGTGGAATAGATGTCACCCTGT-3′, R: 5′-CTTGTGAGCTGTGTCTCCGT-3′), Rankl (F: 5′-CCCATCGGGTTCCCATAAAGT-3′, R: 5′-AGCAAATGTTGGCGTACAGG-3′) and Sod1 (F: 5′-CACTTCGAGCAGAAGGCAAG-3′, R: 5′-CCCCATACTGATGGACGTGG-3′). Each sample was run in triplicate, and fluorescence data were collected at the end of each cycle. Each run ended with a melting curve to ensure amplification specificity (from 60 to 95 °C at a 0.5 °C/10 s rate). Expression levels were calculated using the comparative CT method [i.e., 1/(ΔΔCT), where ΔCT is the difference between CT target and CT reference] after normalization to B2m expression level, and relative fluorescence units (RFU) were analyzed using optical system software Version 3.1 (Bio-Rad).