RNA isolation, cDNA preparation.
Total RNA Purification kit (Jena Bioscience, Germany) was used to extract total RNA from EDTA peripheral blood samples after the manufacturer’s protocol. The reverse transcription process was done by the SCRIPT Reverse Transcriptase kit (Jena Bioscience, Germany) producing the first-strand cDNA using Oligo (dT) primer and M-MLV RT enzyme. In ice, we mix 1.5 μl RNase-free water, 10 μl RNA template and 1 μl Oligo-(dT) primer together, then 4 μl SCRIPT RT buffer, 1 μl dNTP Mix, 1 μl RNase inhibitor, 1 μl Dithiothreitol stock solution and 0.5 μl SCRIPT reverse transcriptase were added then incubated at 30°C for 10 min and 50°C for 60 min. The cDNA was stored at -20°C until analysis.