At the end of the isolation process, the concentration of samples was determined with a spectrophotometer (Nanodrop; Thermo Fisher Scientific, Wilmington, DE, USA).
RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for cDNA synthesis. Reverse transcriptionquantitative
polymerase chain reaction (RTqPCR) was performed using ABI Biosystems™ StepOne™ and RealQ Plus 2x Master Mix Green (Ampliqon A/S, Odense, Denmark).
The β2M housekeeping gene was used as the internal control of qPCR reactions. Reactions were amplified in a thermal cycler
(Applied Biosystems, Thermo Fisher Scientific Inc., USA) with thermal conditions set at 94 °C for 10 min followed by 40 cycles of 15 second
at 94 °C, 60 second at 58 °C, a final extension of 7 min at 72 °C, and melt curve analysis of 55~95 °C at 0.5 °C per five second.
The analysis of real-time PCR data was performed using the 2-ΔΔCt method 16 (link)with target mRNA expression in each sample normalized against the endogenous control.