*2.6. Quantitative PCR*

The gene expression analysis of *HMOX1*, *KRT19*, *LDHA*, *HSPD1*, *MAPK1*, and *CA2* in lesional and healthy skin was performed using the method of quantitative PCR (qPCR). The Qiagen RNeasy Mini Kit with spin columns was used to isolate the total RNA from the skin. The isolated total RNA was treated with DNase (Qiagen, Hilden, Germany) to remove the traces of genomic DNA. RNA concentration was measured with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, USA). The M-MLV kit (Promega, Madison, WI, USA) was used for the reverse transcription with oligo-dT (DNA-Synthes, Moscow, Russia) primers according to the manufacturer's protocol.

The primers used in the qPCR experiments (Table 2) were designed in Primer blast (NCBI, USA), checked with the Multiple primer analyzer (Thermo Fisher Scientific, Waltham, USA) for the formation of potential secondary structures and dimers, and synthesized by DNA-Synthes (Moscow, Russia). The experiments were performed in the CFX96 Touch real-time DNA detection system (Bio-Rad, Hercules, CA, USA) using the SYBR-Green master mix supplied by Evrogen (Moscow, Russia) according to the manufacturer's instructions. The following conditions were used to amplify the DNA: 4 min at 95 ◦C, followed by 40 cycles of consequent incubations at 94 ◦C for 15 s and 60 ◦C for 30 s. Each reaction was run in triplicates. 18S RNA was used as a housekeeping gene to normalize the expression levels of the target genes.

**Table 2.** Gene-specific primers used in the qPCR experiments.


The results were analyzed using the standard 2−∆∆CT method [16] to compare the levels of expressed genes. Each ∆Ct value was calculated as ∆Ct = Ct (tested gene) − Ct (housekeeping gene). ∆∆Ct was calculated as ∆∆Ct = ∆Ct (sample of psoriatic patient) − ∆Ct (sample of healthy individual). The experiments were repeated three times for each sample.
