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.