Total RNA was extracted by TRIzol extraction method (Invitrogen), according to the manufacturer's instructions. Equal amounts of RNA (2 μg) were reverse-transcribed into cDNA using the Transcriptor First-Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA). The primers were synthesized by Sigma-Aldrich (St. Louis, MO, USA) for the following genes (Table 1 ).
All samples were tested in triplicate [3 (link)]. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed on a mixture containing 10 μL PCR Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 1 μL forward and reverse primers (Sangon, Beijing, China), 1 μL template DNA, and 8 μL distilled water. The qPCR conditions were as follows: one cycle of initial denaturation (94°C for 3 min), 30 cycles of amplification (94°C for 30 s, 57°C for 30 s, and 72°C for 25 s), one cycle of melting curve measurement (95°C for 5 s, 65°C for 60 s, and a gradual increase in temperature to 97°C), and a cooling period (40°C for 30 s). The data presented were mRNA levels normalized relative to β-actin.
All samples were tested in triplicate [3 (link)]. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed on a mixture containing 10 μL PCR Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 1 μL forward and reverse primers (Sangon, Beijing, China), 1 μL template DNA, and 8 μL distilled water. The qPCR conditions were as follows: one cycle of initial denaturation (94°C for 3 min), 30 cycles of amplification (94°C for 30 s, 57°C for 30 s, and 72°C for 25 s), one cycle of melting curve measurement (95°C for 5 s, 65°C for 60 s, and a gradual increase in temperature to 97°C), and a cooling period (40°C for 30 s). The data presented were mRNA levels normalized relative to β-actin.