{Reference Type}: Journal Article
{Title}: Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA-sequencing.
{Author}: Salik D;El Kaderi Y;Hans C;Lefort A;Libert F;Smits G;
{Journal}: Exp Dermatol
{Volume}: 31
{Issue}: 11
{Year}: 11 2022
{Factor}: 4.511
{DOI}: 10.1111/exd.14652
{Abstract}: Keratinocyte culture is a standard method used to study gene expression, cell differentiation and proliferation. Numerous protocols exist, however their application is frequently unsuitable for small specimens, such as 4-mm punch skin biopsies.<BR>This study compared 3 different methods of keratinocyte culture from paediatric skin biopsies to evaluate which one ensures adequate cell growth for RNA extraction and sequencing.<BR>Thirty-six skin samples were obtained from 4-mm punch skin biopsies from residual human body material from healthy children. They were cultured in vitro according to 3 different methods: enzymatic method, epidermis explant and direct explant method. Keratinocytes were characterized by immunocytochemistry using pan-cytokeratin. RNA extraction was performed with RNeasy Mini kit. Quantity and quality of the extracted RNA was assessed to meet the requirements of library preparation for sequencing.<BR>The direct explant method had largely shown its superiority over the two other methods, with a 100% success rate and an average of 15 days of culture. RNA extraction yielded a mean of 8545.85 ng of RNA per sample with an RQN of 10. Cover-clip immunochemistry staining with pan-cytokeratin had confirmed the absence of fibroblast contamination.<BR>Although the enzymatic method is the most frequently used for keratinocyte culture, it is not suitable small samples required in dermatology. The direct explant method guarantees a high growth rate and the extraction of high quality RNA. Variation in the amount of RNA harvested are related to inter- and intra-individual variations and to the conditions of the experiment.<BR>This study allowed to conclude that the direct explant method is the most efficient and easy method to ensure cell growth when the samples are from 4-mm punch skin biopsies. This technique avoids fibroblasts contamination and obtains a sufficient quantity and quality of RNA to sequence it.