How does the fluorescence quantitative PCR instrument achieve accurate measurement of gene expression levels?
Publish Time: 2025-04-29
The fluorescence quantitative PCR instrument is a highly sensitive and accurate technology that is widely used to measure gene expression levels. By real-time monitoring of the fluorescence signal generated during PCR amplification, qPCR can provide information about the relative or absolute amount of the target gene in a specific sample.1. Basic PrincipleThe basic principle of fluorescence quantitative PCR is based on the traditional polymerase chain reaction (PCR), but a fluorescent marker is introduced in each cycle to monitor the process of DNA amplification in real time. There are usually two main methods for detecting fluorescent signals: the intercalation dye method and the probe method. Intercalation dyes such as SYBR Green directly bind to double-stranded DNA and emit fluorescence; while the probe method uses a specific fluorescently labeled oligonucleotide probe to release a fluorescent signal when the target sequence is amplified. Both methods can provide real-time data on the accumulation of PCR products.2. Establishment of standard curveIn order to accurately measure gene expression levels, it is usually necessary to establish a standard curve. This involves making a series of dilutions using a known concentration of the target DNA template and performing qPCR analysis on it. By plotting the relationship between the Ct value (threshold cycle number, i.e. the number of cycles required for the fluorescence signal to reach the set threshold) and the template concentration, a standard curve can be obtained. This curve allows researchers to infer the corresponding gene copy number or expression level based on the Ct value of the unknown sample.3. Selection of internal reference genesIn practical applications, in order to correct the errors caused by differences in extraction efficiency or other factors between different samples, one or more internal reference genes are often selected as controls. These internal reference genes should be stably expressed in all samples to be tested, such as housekeeping genes such as β-actin or GAPDH. By performing qPCR analysis on the target gene and the internal reference gene at the same time and calculating the ratio between them, more reliable gene expression level data can be obtained.4. Relative quantification and absolute quantificationFluorescence quantitative PCR instrument quantitatively compares the expression changes of the target gene relative to the internal reference gene under different conditions or between different samples; while absolute quantification determines the specific copy number of the target gene. Both have advantages and disadvantages, and which method to choose depends on the purpose of the experiment and available resources.5. Application of data analysis softwareModern fluorescence quantitative PCR instruments are usually equipped with special data analysis software to help users automate the processing of experimental data. These software can not only automatically generate standard curves and calculate Ct values, but also perform statistical analysis, generate reports and other functions. In addition, some advanced software also provides a graphical interface, making the visualization of results more intuitive and easy to understand, greatly improving scientific research efficiency.6. Experimental design and optimizationIn order to ensure the accuracy of fluorescence quantitative PCR results, reasonable experimental design is essential. First, primers should be carefully designed to ensure their specificity and amplification efficiency. Secondly, PCR reaction conditions, including annealing temperature, extension time and other parameters, should be optimized to obtain the best amplification effect. Finally, the experimental operation process should be strictly controlled to reduce the possibility of human error.In summary, fluorescence quantitative PCR instrument achieves accurate measurement of gene expression levels by real-time monitoring of the fluorescence signal generated during PCR amplification. Whether using the insertion dye method or the probe method, with the help of standard curves, internal reference genes and advanced data analysis tools, researchers can obtain reliable and meaningful results.
