*4.5. Processing of Samples*

Homogenized cell lysates were prepared immediately after collection of whole peripheral blood samples (EDTA tubes, 200 μL) using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany, no: 52304).

Total RNA was extracted from homogenized cell lysates stored at −80 ◦C using a mirVana microRNA Isolation kit (Ambion, Austin, USA, no: AM1560) and followed by an enrichment procedure for small RNAs. To minimize DNA contamination, the eluted RNA was treated for 30 min at 37 ◦C with 5 μL of DNase I (Thermo Fisher Scientific, CA, USA, no: EN0521). A RNA fraction highly enriched in short RNAs (<200 nt) was obtained. The concentration and quality of RNA was assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). If the A(260/280) absorbance ratio of isolated RNA was 1.8–2.0 and the A(260/230) absorbance ratio was greater than 1.6, the RNA fraction was pure and used for the consecutive analysis.
