Interleukin-18 expression and the response to treatment in patients with psoriasis

Rasmy, Hanaa; Mikhael, Nancy; Ismail, Somaia

Real-time quantitative PCR was performed using the Applied Biosystems Perkin Elmer 7300 sequence detection system. Primers were IL-18 sense primer 5'-AGG AAT AAA GAT GGC TGC TGA AC-3' and antisense primer 5'-GCT CAC CAC AAC CTC TAC CTC C-3'. Each PCR reaction (in a 50 μl volume) contained 1× of TaqMan universal PCR master mix, (2×) 50 nM to 900 nM of forward primer, 50 nM to 900 nM of reverse primer, 250 nM of TaqMan probe labelled with 6-carboxyfluorescein (FAM) and 10 ng to 100 ng of cDNA. The amplification protocol was 2 min at 50°C, 10 min at 95°C, then 40 cycles for 15 s at 95°C and 1 min at 60°C. Relative gene ex pres sion was determined for each sample. Amplification of the gene for glyceraldehydes-3phosphate dehydro genase (GAPDH), a constitutively expressed housekeeping gene, was performed on all samples. All genes were subsequently normalized against GAPDH levels. In this study mRNA levels were expressed in picograms/microlitre (pg/μl).