|1||Total RNA extraction.|
|2||Confirm the good purity and quantity of RNA, with 260/280 nm > 1.8 and >200 ng yield by NanoDrop spectrophotometer.|
|3||cDNA strand Synthation using cDNA kit|
|4||QPCR primer design using premier 5.0 for respective genes and confirmation of their accuracy through bioinformatics UCSC software.|
The QPCR of the cDNA was performed by using master mix probe and the previously designed and checked primers. The protocol was as follows:|
95 °C for 5 min, denaturing cycles as 94 °C for 30 s, annealing as 30-s temperature (Tm), extending at 30 s for 72 °C, with a final extension of 10 min at 72 °C.
|6||Control group in experiment as after normalization of mRNA level and as a control group of actin.|
|7||The experiment samples were conducted in three replicates.|