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To prevent mis-hybridization, it is particularly important during the First Strand cDNA Synthesis that the
RNA / FS1 mix is stably kept at 42°C, after the denaturation step (steps 2, 3, and 4 of the protocol, in the user guide), and that the FS2 / E1 mastermix is also pre-warmed and kept at this same temperature (42°C) until is added to the samples (step3step 3).
If the alternative protocol for low input/degraded samples is used, and step 2 is skipped, the
F1 / F2 / E1 mastermix should be pre-warmed at 42°C before being added to the RNA samples (step 3) and the thermocycler should also be pre-heated at 42°C before the samples are transferred on it (step 4).
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