The cDNA synthesis from mRNA was carried out with TaqMan Reverse Transcription reagents (Thermo Fisher Scientific, Waltham, MA, USA). First, 250 ng mRNA of each sample was pre-incubated with 5 µL random hexamer at 85 °C for 3 min. Next, 10 μL 10× PCR buffer, 22 μL MgCl2, 20 μL deoxyribonucleoside triphosphate, 6.25 μL multiscribe reverse transcriptase, and 2 μL RNase inhibitor was added per sample. Lastly, reaction was performed under these conditions: annealing (25 °C, 10 min), reverse transcription (48 °C, 60 min), and enzyme inactivation (95 °C, 5 min).
For miRNA, reverse transcription was performed with the miRCURY LNA RT Kit (Qiagen, Hilden, Germany). For each sample, 10 ng of RNA was mixed with 2 μL of 5x reaction buffer, 5 μL of nuclease free water, and 1 μL of enzyme mix. Reaction was performed using the following program: reverse transcription (42 °C, 60 min) and enzyme deactivation (95 °C, 5 min).
Reactions were carried out on a PRISM 7700 Cycler (Applied Biosystems, Waltham, MA, USA) and transcribed cDNA was stored at −20 °C until further analysis.
For miRNA, reverse transcription was performed with the miRCURY LNA RT Kit (Qiagen, Hilden, Germany). For each sample, 10 ng of RNA was mixed with 2 μL of 5x reaction buffer, 5 μL of nuclease free water, and 1 μL of enzyme mix. Reaction was performed using the following program: reverse transcription (42 °C, 60 min) and enzyme deactivation (95 °C, 5 min).
Reactions were carried out on a PRISM 7700 Cycler (Applied Biosystems, Waltham, MA, USA) and transcribed cDNA was stored at −20 °C until further analysis.