The exact primer concentration and reaction temperatures (up to 50 °C for first strand synthesis and second strand synthesis) strongly depend on the custom primer(s) used. These need to be optimized specifically for each set of custom primers.
NOTE: The higher the primer concentration the higher the likelihood of non-specific priming.
First Strand Synthesis
The final concentration of a target-specific reverse transcription primer should be 12.5 nM – 1.25 µM in the first strand synthesis reaction.
To achieve this concentration rage, you would add e.g., 5 µl of a 50 nM – 5 µM target-specific primer to the reaction.
ATTENTION! The total concentration of oligos in the first strand synthesis reaction should not exceed 2 µM.
Second Strand Synthesis
The final concentration of a target-specific second strand synthesis primer should be 0.5 µM (i.e., 2 µl of 7.5 pM Custom Target Primer).
ATTENTION! The total concentration of all oligos in the second strand synthesis reaction should not exceed 2 µM.
The annealing temperature should be chosen according to the Tm of the targeted primers and can range from 45 °- 72 °C. The extension temperature should be 72 °C.