Annealing Temperature
 

Kindly note that I have three genes with the below listed Tm and I would like to use RT-PCR SYBR Green application method. What is the best Annealing Temperature may I have to use to get good results. ?

Primer F Gene 1 Tm: 53oC
Primer R Gene 1 Tm: 54oC
Primer F Gene 2 Tm: 53oC
Primer R Gene 2 Tm: 52oC
Primer F Gene 3 Tm: 55oC (GAPDH)
Primer R Gene 3 Tm: 49oC (GAPDH)

Thanks
Masood