3.3. Measurement of Cytokine Expression by qRT-PCR

Macrophages were incubated for 2 or 4 h with 30 nM rC5a as indicated. After incubation, macrophages were washed with PBS and total RNA was isolated using a RNeasy kit with on-column DNase I digestion (Qiagen, Germantown, MD, USA). RNA samples were quantified with a ND-1000 spectrophotometer and 65–80 ng of total RNA from macrophages was reverse-transcribed using the iScript cDNA synthesis kit (Biorad, Hercules, CA, USA). For quantitative RT-PCR analyses, primers for human GAPDH, TNF-Alpha, DKK2, FOS, GPNMB, and CAT were used (Supplemental Table S2). An Applied Biosystems 7500 Real-Time PCR system was used with Power SYBR Green Master mix (Applied Biosystems, Waltham, MA, USA) at an amplification profile of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The specificity of all reactions was determined by melting curve analyses at the end of PCR cycles. Gene expression was calculated using the minus delta–delta Ct method (2^−ΔΔCt) method by normalizing the threshold cycle (Ct) values against the reference gene GAPDH [67].