The fluorescence quantitative PCR analyzer achieves precise gene quantification by real-time monitoring of fluorescence signal changes, combined with Ct values and standard curve analysis. The core principle and implementation steps are as follows:

core principle

Add fluorescent groups (such as SYBRGreen dye or TaqMan probe) to the PCR amplification reaction system to synchronize the fluorescence signal intensity with the yield of the amplified product. After completing each PCR cycle, the instrument collects a fluorescence signal and generates an amplification curve. By setting a fluorescence threshold (usually 10 times the standard deviation of the baseline signal), determine the number of cycles (Ct value) at which the amplification product reaches the threshold. The Ct value is negatively correlated with the initial template quantity, meaning that the more initial templates there are, the smaller the Ct value.

Implementation steps

Standard curve construction: Gradient dilution is performed using standard samples of known concentrations (such as purified plasmid DNA or in vitro transcribed RNA), and PCR amplification is performed using different concentrations of standard samples as templates. Draw a standard curve with the logarithm of the standard copy number as the x-axis and Ct value as the y-axis. When the concentration range of standard samples is between 10 ² -10 ¹⁰ copy numbers, the template concentration usually shows a good linear relationship with Ct values (correlation coefficient R ²>0.99), ensuring accurate quantification within this range.

Unknown sample detection: Simultaneously amplify the unknown sample with the standard sample, substitute its Ct value into the standard curve formula, and calculate the copy number of the target gene in the sample. For example, if the standard curve formula is y=3.1372x+38.201 (y is Ct value, x is copy number logarithm), the initial template size of the sample can be obtained by reverse calculation.

Internal reference gene correction (relative quantification): When comparing gene expression differences between different samples, housekeeping genes (such as GAPDH, β - actin) are selected as internal references. The Ct value difference (Δ Ct) between the target gene and the internal reference gene is calculated, and the relative expression level is further analyzed using the Δ Ct method to eliminate the influence of sample size and reaction efficiency differences.