RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from 34 blood samples using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Genomic DNA was removed from the RNA samples using RNase-free DNase I (Fermentas Life Sciences, Thermo Fisher Scientific, MA, USA). One microgram of the Total RNA was used to generate a single strand of cDNA using the QuantiTect Reverse Transcription Kit (Qiagen TM, Germany), according to the manufacturer’s instructions. PCR was performed on samples with and without reverse transcriptase (RT) added to the reaction mix, using the primers reported in Table 1. The conditions used for PCR were: 94 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. All the amplicons were sequenced.