AAT Bioquest

What is the workflow of real-time PCR?

Posted May 12, 2023


Real-time PCR involves the conversion of RNA to cDNA (complementary DNA) via reverse transcription. This is followed by multiple PCR cycles to amplify and detect the genes of interest. The products are detected in real time using fluorescent reporters, most commonly SYBR green and Taqman probes. 

The workflow of real-time PCR can be summarized as follows: 

Unique or custom primers are added to PCR reagents to set up amplification reactions.

These reactions are then run in specialized thermal cyclers equipped with fluorescence detection modules. The fluorescent reporter molecule included in each reaction well yields increased fluorescence corresponding to an increasing amount of product DNA

As amplification occurs, the fluorescence signal is monitored by the fluorescence detection modules in the thermal cycler. The measured fluorescence is proportional to the amount of PCR product. 

The amount of amplicon produced in each cycle is calculated using the change in fluorescence over time.  The collected data is then analyzed using proprietary instrument software. 

Additional resources

Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression

Real-Time PCR (qPCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*