Total RNA was isolated with a kit (RNeasy Plus Mini Kit; Qiagen Inc., Valencia, CA) according to the manufacturer’s specifications, and real-time quantitative reverse-transcription–polymerase chain reaction (qPCR) was performed as we have previously described.
28 30 Briefly, duplicate reactions were prepared with 18 μL PCR master mix consisting of 9 μL master mix (iQ SYBR Green Supermix; Bio-Rad, Hercules, CA), 1 μL cDNA template, 1 μL each of gene-specific primer pairs
(Table 1) , and 6 μL RNase-free water. Reactions were denatured at 95°C for 2 minutes and amplified for 50 cycles at 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. Real-time quantification of mCRP genes was normalized to the threshold cycle (C
T) value of either human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or mouse peptidyl-prolyl
cis-trans isomerase A in the corresponding species, where C
T equals the PCR cycle number at which the amount of amplified sample product reached 100 relative fluorescence units. Fold difference of mCRP expression, relative to HeLa cells, nontreated cells, or mouse testis expression, was calculated by comparing C
T (2
−ΔΔCT ).
30 A melting curve for all products was obtained immediately after amplification by increasing the temperature in 0.4°C increments from 65° for 85 cycles of 10 seconds each. The experiments were separately repeated three times with similar results.