Sample collection and storage

Collecting domestic pig skin applying method described below does not require an ethical approval. In case of human subjects, the Ethical Review Committee on Human Research of the University of Tartu approved the study protocols and informed consent forms. All participants signed written informed consent.

Domestic pig (mixed from multiple breeds such as Yorkshire, Landrace and Duroc) skin from the front limbs of three female piglets was collected immediately after slaughter. The pig samples were collected free of charge in Tartu County (Estonia) slaughterhouse owned by OÜ Rotaks-R. The skin was cut so that it contained the layers of epidermis, dermis and minimum amount of subcutis. Four different sample collection/storage strategies were tested – samples were (1) collected as dry biopsies and immediately frozen on dry ice; (2) collected into Allprotect Tissue Reagent (APTR; Qiagen, Hilden, Germany), kept overnight at +4 °C and then transferred to −80 °C; (3) collected into QIAzol Lysis reagent (QIAzol) or (4) into RTL lysis buffer from RNeasy Fibrous Tissue Mini kit, containing beta-mercaptoethanol (BME + LB) (both from Qiagen, Hilden, Germany) and frozen at −80 °C 2–3 hours after collection.

All six human subjects in the study were Caucasians living in Estonia and were recruited from among the patients at the dermatologic outpatient clinic. The skin tissues collected for the study were derived from the edges of the unpigmented skin areas from a birthmark removal surgery (two pieces per patient). Among six patients there were two men and four women, the average age was 43 years (±SD 13) and the body areas were abdomen, thigh, chest, arm, and side of a trunk. For human, only two sample collection and storage strategies were used – samples were collected into QIAzol or BME + LB, frozen at −20 °C ~1 hour after collection and transferred to −80 °C within a week after collection (best possible conditions available in clinic).

All domestic pig and human samples were stored at −80 °C for two months before RNA extraction. All the skin pieces were weighed prior homogenizing and for the following procedures full biopsies were used. Each workflow had three biological replicates.