4.2. Total RNA Extraction

Following pulverization of fresh-frozen BCC and SCC cancer and matched healthy-tissue (control) specimens, total RNA was extracted using the TRI Reagent^® (Molecular Research Center Inc., Ohio, USA), following manufacturer’s instructions. RNA pellet was dissolved in RNA Storage Solution (Ambion—Life Technologies—Thermo Fisher Scientific, Waltham, MA, USA) and appropriately stored at −80 °C, until further processing. Total RNA concentration was evaluated by absorbance measurement at 260 nm, in a BioSpec-nano UV–Vis Spectrophotometer (Shimadzu Corp., Kyoto, Japan). RNA structural integrity was visually confirmed by agarose gel electrophoresis. Archival melanoma samples [138], kept in RNAlater RNA stabilization reagent (Qiagen, Redwood City, CA, USA), were released from the reagent, and approximately 20 mg of tissue per sample were disrupted and homogenized. Total RNA was extracted from each melanoma specimen using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. Purified RNA preparations were stored at −80 °C in RNase-free ddH₂O. RNA concentration was determined by measuring the absorbance at 260 nm in a SmartSpec™ Plus Spectrophotometer (Bio-Rad, Hercules, CA, USA).