There are several things that may improve yields: Check the primer design using computer software. Optimize the annealing temperature in a 1-2°C step. A primer concentration of 0.2 μM is satisfactory for most PCR reactions. However, sensitivity and yield of RT-PCR reactions can be improved by increasing the primer concentration to above 0.5 μM. Lower primer concentration between 0.07 μM to 0.2 μM may improve specificity. Increase cycling numbers up to 45 cycles. Do a manual hot-start. Use thin-wall 0.2 ml PCR tubes. Try a touch-down PCR protocol (4).