Quantification of AK2 Gene Expression

Total RNA was reversely transcribed using a PrimeScript RT Mix (Takara, China) in accordance with the manufacturer’s instructions. Following RT-PCR amplification, melting curve analysis was executed to identify the specific generation of the PCR product with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as internal controls. Fold changes for aberrant expression were calculated using the 2–ΔΔCt method. Reactions were performed in 20 μL system including SYBR Select Master Mix(Thermo Fisher Scientific) and 40 ng cDNA with the following reaction conditions: 95 °C 10 minute, 40 cyclesof 95 °C 30 sec, 58 °C 1 minute, 72 °C 1 minute. The primer used for the amplification of the human AK2 gene was as follows: forward primer, 5′-GCAGAACCCGAGTATCCTAAAGG, and reverse primer, 5′-TTCCCAGCATCCATAGTTGCC. GAPDHwas used as the housekeeping gene with forward primer 5′-TTCGACAGTCAGCCGCATCTTCTT and reverseprimer 5′-ACCAAATCCGTTGACTCCGACCTT. Delta-delta Ct values were used to determine their relative expression as fold changes, as previously described^{24 (link)}.