PEER-REVIEW | DERMATOLOGY | Table 1 Patient characteristics


PATIENTS WITH PSORIASIS


NUMBER OF PATIENTS


SEX MEAN AGE 10


PATIENTS WITH ISCHEMIC HEART DISEASE AND ATHEROSCLEROSIS OF PERIPHERAL ARTERIES


10


Male Female Male Female 55 5 54.22.4


5 61.31.5 samples from


unaffected skin regions were taken at a


Psoriasis For the quantitative determination of the changes in ribonucleic acid (RNA) expression, the biopsy material used was collected from 10 patients with psoriasis. The mean age was 54.2 years (Table 2). Skin sample collection was performed under local anesthetic with the use of dermatological biopsy punch (4 mm). Patients received no systematic PUVA/UV therapy for a month before the skin biopsy. Biopsy samples from unaffected skin regions were taken at a distance of 3–4 cm from psoriatic lesions. RNA isolation was performed


Biopsy


distance of 3–4 cm from psoriatic lesions.


from biopsies using Qiagen columns and standard RNeasy Mini Kit skin protocols (Qiagen, Germany). To remove DNA contamination from RNA


preparations they were treated with Qiagen DNase. A reverse transcription reaction was performed as


follows. In 200 l PCR tube buffer, add deoxynucleotide (dNTP), 100 U Moloney-Murine Leukemia Virus (M–MLV) reverse transcriptase (Promega Corporation, Madison, Wisconsin, USA), 20 U RNasin (RNase inhibitor), 500 ng oligo(dT)12–18 primer, and 100 ng/l of RNA. The mixture was incubated for 1 hour at 37°C.


Table 2 Patients with psoriasis diagnosis


PATIENT AGE SEX DIAGNOSIS 160


241 336 436 569 665 757 849 952


10 36 


F Psoriasis vulgaris, progressive stage. Psoriatic arthritis M Psoriatic arthritis, psoriatic erythroderma M Psoriasis vulgaris, exudative type M Psoriatic erythroderma


M Psoriasis vulgaris, exudative type M Psoriasis vulgaris, exudative type


F Psoriasis vulgaris, exudative type. Psoriatic arthritis M Psoriasis vulgaris, exudative type. Progressive stage F Psoriasis vulgaris, progressive stage. Psoriatic arthritis 77 M Psoriasis vulgaris, exudative type. Progressive stage


August/September 2015 | prime-journal.com


Real-time PCR was conducted in 96-well optical plates using fluorescence labeled oligonucleotide probes. Reaction was conducted using a reaction mixture 2.5 times the volume of the total PCR mixture, supplemented with ROX reference dye (Syntol, Russia). Primers and probes were obtained from DNA Synthesis. Amplification was conducted using a thermal cycler


(iQ4, Bio-Rad Laboratories, Hemel Hempstead, UK) as follows:  Denaturation at 95°C for 4 minutes  Denaturation at 94°C for 15 seconds  Annealing at 58°C for 15 seconds  Synthesis at 72°C for 15 seconds  Steps 2–4 were repeated 35 times. GAPDH was used as a housekeeping gene and target


gene expression was normalized in respect to its messenger RNA (mRNA) quantity. GAPDH and target gene expression was conducted in separate tubes. Analysis of the PCR results was conducted using the 2-CT method, according to Livak and Schmittgen7


.


Ct was calculated as follows: Ct = Ct(affected biopsies) – Ct(unaffected biopsies), with Ct = Ct(the analyzed gene) – Ct(GAPDH). Each sample was analyzed in triplicate.


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