Tetro reverse transcriptase enzyme

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.

Total RNA was extracted from glioma specimens using TRIzol reagent (Invitrogen, Massy, France), as detailed previously [64 (link),65 (link),66 ]. RNA concentration was determined using a NanoVueTM Plus spectrophotometer (GE Healthcare, Hatfield, UK). We then diluted the samples with ultrapure water, in order to have same concentration of RNA per tube. In accordance with the instructions of the manufacturer, cDNA was initially synthesized using the Tetro reverse transcriptase enzyme (Bioline, Livron-sur-Drôme, France) from 0.5 μg of total RNA in a 20 μL reaction mixture with 1 μL of 25 µg random hexamer primer (Bioline, France) and 4 μL of RNase-free water was added and incubated at 70 °C for 5 min. After that, 4 μL of dNTP (10 mM), 4 μL of Tetro reverse transcriptase buffer, 0.5 μL of Tetro reverse transcriptase enzyme (Bioline, France), 0.5 μL of RNase inhibitor (Invitrogen, France), and 1 μL of RNase-free water were added and then incubated at 25 °C for 10 min, followed by 45 °C for 30 min, and finally 85 °C for 5 min.

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