2.2. RNA Extraction and cDNA Synthesis

Total RNA extraction, DNase digestion and cDNA synthesis from all the samples were performed as previously reported [9,32]. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) from 1 × 2-cm square pieces of tissue paper, 30 μL of fluid or 5 × 5-mm square pieces of swab head. Possibly contaminated DNA was digested using an RNase-Free DNase Set (Qiagen, Germantown, MD, USA). An aliquot of 5 μL of DNA-free total RNA was added to 10 μL of the reverse transcription mixture of a Primescript RT Reagent Kit (Takara Bio, Otsu, Japan). All procedures were performed according to the manufacturer’s instructions. Successful removal of DNA contaminants was confirmed using no-reverse transcription controls of representative samples for each type of body fluid.