In molecular biology, real-time quantitative PCR (qPCR) is undoubtedly a revolutionary technology. It enables the detection and quantification of DNA or RNA sequences and serves as a precise tool for gene expression analysis, pathogen diagnosis, and other applications.
However, a critical factor in qPCR experiments is often overlooked—the Threshold Line setting.
Today, we will unveil the mystery of how adjusting the qPCR threshold line impacts the Cycle Threshold (Ct) value.
During the qPCR amplification process, fluorescence signals gradually increase as the number of cycles progresses. The threshold line is a manually set cutoff value for fluorescence signals. It can be compared to defining a passing score in an exam—if 60 is set as the passing mark, scores above 60 pass, while those below 60 fail. Similarly, the threshold line is a predefined fluorescence signal level that determines when a sample is considered to have a significant amplification signal.
Four Phases of the Amplification Curve: Baseline Phase, Exponential Phase, Linear Phase, and Plateau Phase
In qPCR, instruments and reagents inherently generate background fluorescence signals, known as the baseline. The threshold line is set above this background noise to distinguish genuine amplification signals from background fluorescence.
The Ct value is the cycle number at which the fluorescence signal first exceeds the threshold line. A lower Ct value indicates a higher initial concentration of the target template and vice versa.
Raising the Threshold Line:
If you move the threshold line upward, a stronger fluorescence signal is required to reach the threshold, resulting in a higher Ct value. This means the sample needs more cycles to accumulate sufficient fluorescence intensity, potentially underestimating the initial concentration of the target gene.
Lowering the Threshold Line:
Conversely, if the threshold line is lowered, fluorescence signals will cross the threshold earlier, leading to a lower Ct value. This may overestimate the initial concentration of the target gene.
Where should the threshold line be set? Proper threshold line setting is critical for obtaining accurate and reliable qPCR results.
Principles for Settingthe Threshold Line:
1. The threshold should be above background fluorescence.
• The threshold must be set above the background fluorescence levels of the samples and the highest fluorescence value of negative controls to ensure that only true amplification signals are detected, avoiding background noise interference.
2. The threshold should be set within the exponential phase.
• The threshold should ideally be placed within the exponential phase of the amplification curve. This is because the fluorescence signal grows exponentially in this phase, making it the most stable and reproducible stage for qPCR measurements. Selecting this phase ensures higher accuracy and reliability of results.
3. Avoid setting the threshold too high or too low.
• A low threshold may lead to interference from noise signals, reducing accuracy.
• A high threshold may fall into the linear or plateau phase, where fluorescence signal changes become unpredictable, making results difficult to interpret.
Therefore, the threshold line should be set within a reasonable range—above baseline noise but not too close to the plateau phase. Generally, the fluorescence threshold is set within the exponential phase of PCR amplification and corresponds to 10 times the standard deviation of fluorescence signals during the initial 3–15 cycles of PCR.
For consistency and comparability of experimental data, it is recommended to use the same threshold setting within the same experiment and maintain consistent settings across different batches whenever possible.
The LongGene OPTIMAL qPCR analysis software automatically sets the threshold line by default. When viewing the results, the software intelligently determines the optimal threshold line for the experiment.LongGene PCR equipment
• The software sets a separate threshold line for each target gene, as different targets have distinct optimal thresholds, and well positions also influence threshold determination.
• Users can manually adjust the threshold using the software interface. Adjusting the threshold line up or down will directly affect the corresponding Ct values.
As long as the threshold is set within a reasonable range, the relative Ct values (ΔCt) between two samples will remain unchanged. This allows for accurate fold-change calculations in gene expression and absolute quantification using standard curves.
A thorough understanding of qPCR threshold line adjustments allows for better control of experimental variables, enhancing the precision and reliability of results.
