Table 1. Comparison of run time and time saved between standard PCR and optimized Fast RT-PCR protocols.

Protocol	Stages	Temperature and time
		QuantStudio™ 5 Real-Time PCR System	Applied Biosystems™ 7500 Fast Dx Real-Time PCR System	CFX96 Touch Real-Time PCR detection System
Standard PCR	Reverse transcription	55°C- 10 minutes	55°C- 10 minutes	55°C- 10 minutes
	RT enzyme inactivation	95°C- 1 minute	95°C- 1 minute	95°C- 1 minute
	Polymerase activation & Cycle denaturation	95°C- 10 seconds	95°C- 10 seconds	95°C- 10 seconds
	Combined annealing and extension	60°C- 1 minute	60°C- 1 minute	60°C- 1 minute
Fast PCR	Reverse transcription	55°C- 5 minutes	55°C- 8 minutes	55°C- 8 minutes
	RT enzyme inactivation	95°C- 1 minute	95°C- 1 minute	95°C- 1 minute
	Polymerase activation & Cycle denaturation	95°C- 7 seconds	95°C- 5 seconds	95°C- 5 seconds
	Combined annealing and extension	60°C- 30 seconds	60°C- 30 seconds	60°C- 30 seconds
Standard RT-PCR run time		86	95	103
Fast RT-PCR run time		49	48	61
Time saved		37	47	42

Fast RT-PCR protocol has been optimized so that it gives results which are similar to Standard RT-PCR result in terms of shape of fluorescence intensity curves, PCR efficiency and precision. The protocols have been optimized on QuantStudio™ 5 Real-Time PCR System, Applied Biosystems™ 7500 Fast Dx Real-Time PCR System, CFX96 Touch Real-Time PCR detection System. In addition to the protocols, the last three rows of Table 1 shows total RT-PCR run time for Standard and Fast PCR across the thermal cyclers and time saved (last row) due to Fast PCR amplification.