Automatic Setting
Most real-time qPCR instruments automatically set the fluorescence threshold. Typically, this threshold is determined as 10 times the standard deviation of the baseline signal.
Manual Setting
Users can manually adjust the threshold line, usually placing it within the exponential phase of the amplification curve—where the fluorescence signal shows a significant increase. Manual setting requires experience and precision, and it is commonly done during the mid to late exponential phase.
Principles and Purpose of Threshold Setting
The threshold should be set above the baseline but low enough to fall within the exponential phase of amplification. The purpose is to ensure amplification efficiency is close to 2 (i.e., doubling of DNA with each cycle) while avoiding the linear or plateau phases, which could compromise data accuracy.
Impact of Threshold Setting on Experimental Results
The threshold setting directly affects the Ct (cycle threshold) value. If the threshold is set too high, it may fall within the linear or plateau phase of amplification, where efficiency declines and data variability increases—leading to inaccurate results. Therefore, a properly set threshold is critical for ensuring the accuracy and reproducibility of the experiment.
What is a Threshold?
The threshold is the horizontal line you adjust up or down in the software interface—it plays a key role in determining the Ct value. It represents the point at which the fluorescence signal rises significantly above the baseline. Most instruments calculate this threshold based on the average baseline signal, setting it approximately 10 times higher than the baseline standard deviation.
In other words, if the baseline is properly set, the threshold can also be manually adjusted to an accurate value.
Typical Amplification Curve Phases
A standard amplification curve consists of four distinct phases:
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Baseline Phase
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Exponential Phase
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Linear Phase
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Plateau Phase
Concept of the Threshold Line
The threshold line is a horizontal line that intersects the amplification curve and is used to determine the Cycle Threshold (Ct) value of a fluorescence signal.
Because the Ct value is determined by where the amplification curve crosses this line, the threshold line is extremely important!
Typically, the fluorescence threshold line is set during the exponential phase of PCR amplification. It is generally defined as 10 times the standard deviation of the fluorescence signal observed during the baseline cycles (usually cycles 3 to 15 of the PCR reaction).
Principles for Adjusting the Threshold Line
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Place the threshold line within the mid-to-late exponential phase of the amplification curve, as this region offers the highest reproducibility and best reflects the true starting point of PCR amplification.
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The threshold line must be above the negative control and baseline noise to avoid false positives or interference from background signals.
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The optimal position for the threshold line is where the amplification curve becomes approximately linear and where curves from different samples run parallel to each other.
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In multiplex qPCR experiments, each target or fluorescence channel should have its own appropriately set threshold line, as different targets may exhibit varying signal intensities and exponential phase timing.
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If the threshold is set too high, the resulting Ct value will be larger, potentially falling within the linear or plateau phase of amplification, thus compromising the accuracy of the data.
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If the threshold is set too low, the Ct value may be underestimated and could be affected by background noise, leading to inaccurate or misleading results.
Q9600 Series Real-time Fluorescence Quantitative PCR Instrument
The Q9600 Series is a high-throughput real-time fluorescent quantitative PCR instrument featuring side fluorescence detection channels. All fluorescence channels are collected simultaneously, enabling the detection of 96 samples across all channels in just 5 seconds, significantly reducing analysis time.
Equipped with an automatic tray ejection system, the Q9600 can be seamlessly integrated with automated workstations, enhancing operational efficiency. It supports PC-based operation, allowing a single computer to control multiple instruments and run several experiments concurrently.
The software is powerful and versatile, supporting qualitative and quantitative analysis, high-resolution melting (HRM), and genotyping.
With robust hardware and software capabilities, the Q9600 Series is designed to meet the needs of diverse application scenarios and customer requirements.