Total RNA was extracted from PBMCs and frozen glioma samples using TRIzol reagent (Invitrogen, France) as previously described (24 (link)). RNA concentration and quality were measured using the NanoVueTM Plus Spectrophotometer (GE Healthcare, UK), then cDNA was synthesized using Tetro Reverse Transcriptase Enzyme (Bioline, France) from 0.5 μg of total RNA in a 20 μl of reaction mixture according to the manufacturer’s instructions, mixed with 1 μl of Random Hexamer Primer 25µg (Bioline, France) and 4 μl of RNase-Free water, then incubated at 70°C for 5 min to break the secondary structures of RNA.
Next, 4 μl of Tetro Reverse Transcriptase buffer, 4 μl of dNTP (10 mM), 0.5 μl of RNase Inhibitor (Invitrogen, France), 0.5 μl of Tetro Reverse Transcriptase Enzyme (Bioline, France), and 1 μl of RNase-Free water were added and incubated at 25°C for 10 min then at 45°C for 30 min then at 85°C for 5 min.
Next, 4 μl of Tetro Reverse Transcriptase buffer, 4 μl of dNTP (10 mM), 0.5 μl of RNase Inhibitor (Invitrogen, France), 0.5 μl of Tetro Reverse Transcriptase Enzyme (Bioline, France), and 1 μl of RNase-Free water were added and incubated at 25°C for 10 min then at 45°C for 30 min then at 85°C for 5 min.
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