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First Strand Synthesis
The concentration of a target-specific reverse transcription primer should be around 12.5 nM – 1.25 µM final concentration (in this case it would mean you would need 5 µl of a 50 nM – 5 µM target-specific primer). The total concentration of oligos in the first strand synthesis reaction should not exceed 2 µM. The higher the primer concentration the higher the likelihood of unspecific binding. However, the exact primer concentration and reaction temperature (up to 50 °C for first strand synthesis and second strand synthesis) strongly depends on the custom primer(s) used and has to be optimized accordingly.
Second Strand Synthesis
The concentration of a target-specific second strand synthesis primer should be 0.5 µM final concentration of the target primer (i.e., 2 µl of 7.5 pM Custom Target Primer). The total concentration of all oligos in the second strand synthesis reaction should not exceed 2 µM. The higher the primer concentration, the higher the likelihood of unspecific binding. The annealing temperature should be chosen according to the Tm of the targeted primers and can range from 45 °C to 72 °C. The extension temperature should be 72 °C.