RNA extraction by peripheral blood mononuclear cells (PBMCs) and subsequent retro-transcription were performed using the RNeasy Mini Kit and QuantiTect Reverse Transcription Kit (Qiagen Sciences, Germantown, USA), as previously described [15 (link)]. For mRNA extraction, each whole blood sample (approximately 8 ml) was processed within 2 h of collection and the mRNA was stored at –80°C. Two whole blood samples were processed for each patient.
The quantification of RNA was performed using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). The tracks of genomic DNA were removed using the QuantiTect Reverse Transcription Kit (Qiagen, Germany), with the thermocycler program: 2′ at 42°C with gDNA wipeout buffer. Complementary DNA (cDNA) synthesis was carried out using 100 ng of RNA and the QuantiTect Reverse Transcription Kit (Qiagen Sciences, Germantown, USA), thermocycler program: 15′ at 42°C and 3′ at 95°C.
The quantification of RNA was performed using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). The tracks of genomic DNA were removed using the QuantiTect Reverse Transcription Kit (Qiagen, Germany), with the thermocycler program: 2′ at 42°C with gDNA wipeout buffer. Complementary DNA (cDNA) synthesis was carried out using 100 ng of RNA and the QuantiTect Reverse Transcription Kit (Qiagen Sciences, Germantown, USA), thermocycler program: 15′ at 42°C and 3′ at 95°C.
