Real-time fluorescence quantitative PCR incorporates a fluorescent group into the detection system. By monitoring the fluorescent signal of each product during each cycle of the PCR amplification reaction, quantitative and qualitative analysis of the starting template is achieved. As the PCR reaction proceeds, fluorescent PCR products gradually accumulate, and the signal intensity increases proportionally. This allows us to monitor the amount of PCR product in real time through changes in the fluorescence signal, generating an amplification curve.

A real-time fluorescence quantitative PCR amplification curve has three phases: baseline, exponential, and plateau.
During the baseline phase, the fluorescence signal is essentially background, making it impossible to discern the product's fluorescence value.
During the exponential phase, the fluorescence signal exceeds the background signal and enters the exponential growth phase. The cycle number at this point is called the Ct (Cycle Threshold). The logarithm of the starting PCR template concentration is linearly related to the Ct.
During the plateau phase, the amplified product no longer increases exponentially, and the final PCR product amount cannot be used to calculate the starting template copy number.

Quantification methods include absolute and relative quantification, using the SYBR Green I assay and Taqman probe method.

Absolute quantification: Absolute quantification, also known as the standard curve method, uses a known standard curve to quantify the starting amount of a target template in an unknown sample. Using a standard prepared by a 5-6-point serial dilution (CoBioGene offers a large number of validated standards), amplification is performed via real-time fluorescence quantitative PCR. A standard curve is plotted with the logarithm of the initial copy number of the target template as the horizontal axis and the detected CT value as the vertical axis. A linear regression equation is generated, and the CT value of the unknown sample is inserted into this equation to calculate the starting amount of the target template.

Relative quantification: Relative quantification can analyze the ratio of mRNA expression levels of a target gene between different samples, between different sites in the same sample, and within a site within a sample at different dynamic periods. It can also analyze the copy number ratio of the target gene to that of an internal reference gene in the same sample.

Theoretically, it can detect as few as a few copies of nucleic acid molecules

Especially using the TaqMan probe method, it can effectively distinguish highly homologous sequences and single nucleotide polymorphisms (SNPs).

Provides digital results with a wide dynamic range (spanning 7-8 orders of magnitude).

The 96-well or 384-well plate format allows for simultaneous testing of large numbers of samples, resulting in extremely high efficiency.