What are the disadvantages of single step RT-qPCR?

One limitation is that when RNA quality is lower and inhibitors are unevenly distributed among samples, it can disrupt reaction efficiency to different extents; this lowers the consistency of results across samples. Furthermore, in one-step RT-qPCR, the inability to preserve the cDNA synthesis product means that extra aliquots of the initial RNA sample(s) are necessary to repeat reactions or for observing the expression of different genes. It is also not possible to optimize two reactions individually.