2.5. Synthesis of cDNA
The complementary DNA was synthesized from RNA by using a kit (Revert Aid Reverse Transcriptase, Thermo Scientific, Catalog # EP0442). Briefly 11 uL template RNA were mixed with 1.0 µl of Oligo (dt) 18 primers (#S0132) and incubate for 5 mints at 65°C. Then 4.0 µl of RT buffer, also add 1 uLRibolock Rnase inhibitor and 1 µl of reverse transcriptase (Revert Aid) and 2 µl of dNTPs mix (#R0181) were added to making a total volume of 20 µl. now the mixture was centrifuged briefly and incubated at 42°C for 80 minutes. Then the reaction was Terminate the by heating at 70 °C for 5 minutes cDNA were stored at minus 20 for RT-PCR analysis.