sentence: According to the manufacturer ’ s instructions , the 20 μ l reaction mixture contained 0.5 μ l of each primer , 1.0 μ l of cDNA , 10 μ l of 2 × M5 HiPer Realtime PCR Super mix with Low Rox and 8.0 μ l of ddH O qRT-PCR was performed using the following thermal profile : 95 ° C for 30 s , 40 cycles of 95 ° C for 15 s , 65 ° C for 15 s , and 72 ° C for 30 s. Three technical replicates were used for each gene .