*2.2. RNA Isolation*

RNA isolation from the buffy coat obtained after centrifugation (150 g, 4 ◦C, 10 min.) was performed by the use of a commercial QIAamp® RNA Blood Mini set. Genetic material was isolated from 3.5 mL of peripheral blood obtained from all volunteers using EDTA tubes and the BD Vacutainer® Blood Collection system. The isolation process was fully compliant with the manufacturer's guidelines. The isolation process was extended by an additional purification step using the RNase-Free DNase Set to obtain the purest product free of any genomic DNA. All of the procedures were carried out within no more than 2 h from the collection of a blood sample. Part of extracted RNA was used to visualize a product and obtain cDNA immediately after the isolation process; the rest of the genetic material was stored at −80 ◦C until analyzed.

### *2.3. Spectrophotometric Evaluation of Isolated RNA and Gel Visualization*

At the end of the isolation procedure, 1.5 μL of each sample was pipetted into a sterile Eppendorf tube, which was then placed in an ice block, in order to assess the quality and quantity of RNA obtained. An additional water-containing blank was prepared to calibrate the device (NanoDrop), which was used for RNA elution in the final isolation step. For visualization of RNA, 1.2% agarose gel was prepared based on TAE buffer. Agarose gel was enriched after cooling with 10 μL of 5 mg/mL ethidium bromide. Each sample of RNA in a volume of 3.5 μL was heated to 70 ◦C in a water bath for 1 min. and then cooled on ice for another minute. An equal amount of loading buffer was then added to each of the samples, and the mixture was then loaded to the gel. The GeneRuler Plus DNA Ladder size 100 bp from ThermoFisher was used as the size standard. Electrophoresis was carried out at 90 V for 60 min. Subsequently, to visualize the obtained product, the gel was transferred to a Gel-Doc 2000 apparatus that was connected to a computer with Quantity One software. The analysis of the obtained gel allowed a clear distinction between the two isolated RNA fractions: 18S RNA and 28S RNA.
