What’s the difference between one-step and two-step RT-PCR?

One-Step RT-PCR: 

• The RT (reverse transcription) reaction and the PCR (polymerase chain reaction) are combined and carried out in one step
• Both reactions – the RT step and subsequent amplification step – are performed in the same tube
• A single buffer is optimized for both reactions
• The reverse transcriptase and DNA polymerase are premixed into a single tube
• Advantages include fewer steps, more time-efficient, more consistent results, and reduced risk of contamination by extraneous DNA
• Downsides include reduced sensitivity and the inability to individualize and optimize the two processes – cDNA synthesis and PCR
• Preferred technique for high-throughput applications that require simple and fast analysis

 Two-Step RT-PCR: 

• The RT (reverse transcription) reaction and the PCR (polymerase chain reaction) are carried out sequentially
• The two reactions are carried out in separate tubes
• Two separate buffers are optimized for each step
• cDNA is created first in a separate RT reaction and some of the newly created cDNA is added to the PCR
• Advantages include higher sensitivity, higher efficiency, greater flexibility to choose optimum RT enzymes and DNA polymerases for each reaction, and ability to stock cDNA for future use
• Downsides include higher time commitment and the need for more optimization
• Preferred technique for applications with limited amount of starting material such as single cell analysis