Ct (threshold cycle) is calculated as the intersection point between an amplification curve generated during PCR and a pre-defined threshold line.
Here's a more detailed breakdown:
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Amplification Curve: During PCR (Polymerase Chain Reaction), the amount of amplified DNA (or cDNA in the case of RT-PCR) increases exponentially with each cycle. This increase in fluorescence (due to a fluorescent dye or probe binding to the amplified DNA) is plotted against the cycle number, creating an amplification curve.
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Threshold Line: This is a horizontal line set manually or automatically, intersecting the amplification curves in the exponential phase of the reaction. The threshold should be set above the background noise and within the exponential phase for all the amplification curves being analyzed.
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Ct Value Determination: The cycle number at which the amplification curve crosses the threshold line is defined as the Ct value. A lower Ct value indicates that the target sequence was more abundant at the beginning of the reaction, as it took fewer cycles to reach the threshold. A higher Ct value indicates a lower initial concentration.
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Factors Affecting Ct Value: It's important to remember that many factors beyond the initial target concentration can affect the Ct value, including:
- Reaction efficiency
- Primer design
- Enzyme quality
- Instrument calibration
Because of these factors, Ct values are generally used for relative quantification, comparing the expression of a target gene between different samples, rather than absolute quantification.
In summary, Ct is a relative measure of target concentration in a PCR reaction, determined by the cycle number at which the amplification signal crosses a pre-defined threshold.
