Dear Luke,

Thank you for your interest in our poster, Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ dNTPs. An activation step is not required for RT-PCR as the CleanAmp™ dNTPs are activated by the combination of heat at 47°C and the acidification of Tris Buffer with increasing temperature. This releases enough dNTPs to generate a cDNA during the RT reaction. The slower activation helps decrease off-target reaction during both the RT and PCR steps.

I recommend using the conditions shown in Figure 9 of the poster which I have included below for your convenience. Of note, we recommend using a traditional 3-step thermocyling protocol, as it will provide more consistent performance than a faster 2-step protocol.
RT-PCR Conditions: Buffer (50 mM Tris-HCl; 75 mM KCl; 3 mM MgCl₂), 0.4 mM CleanAmp™ dNTPs, 0.5 µM Forward Primer, 1.0 µM Reverse Primer, 100 U M-MLV (RNase H-) RT, 2.5 U Taq polymerase, 0.032-500 ng Target RNA, 10 mM DTT, 0.4 U/µL RNase Inhibitor, 25 µL reaction.
RT-PCR Cycling Conditions: 47°C (30 min); 94°C (10 min); [94°C (15 sec), 62°C (30 sec), 72°C (1 min)] 40X; 72°C (5 min)

Best regards,
Sabrina Shore, MS