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.