What are the limitations of one-step RT-PCR?

The one-step RT-PCR method involves performing both reactions – RT and PCR – in a single step. One of the limitations of one-step RT-PCR is the inability to individualize and optimize the two processes. The single, common buffer optimized for both reactions in the same tube is more likely to be suboptimal as compared to using separate buffers specifically optimized for each individual enzyme. Combining both steps into one makes it more complicated to troubleshoot, which is another limitation of one-step RT-PCR. The third limitation is that this technique does not provide a stock of cDNA for future use.