How is the threshold cycle in a real-time PCR amplification curve determined?

The threshold cycle, often referred to as the Ct value in real-time PCR, is determined by analyzing the point at which the amplification of the target DNA becomes detectable above the background noise. In this context, one critical aspect is the variability in the fluorescence signal, which is assessed using standard deviation of the relative fluorescence units (RFUs).

When analyzing the amplification curve, the threshold is set at a level above this background noise, typically several standard deviations above the average baseline signal. This helps ensure that the increase in fluorescence is representative of the actual amplification of the target DNA rather than fluctuations caused by experimental error or other nonspecific signals. This method of establishing the threshold based on the average standard deviation provides a more reliable and quantifiable means of determining the threshold cycle compared to other methods that might not accurately reflect true amplification.

Other options, such as determining the threshold based on the background signal itself, drawing from maximum fluorescence, or using melt temperature, do not provide the necessary specificity or reliability required in real-time PCR analysis. These approaches may not accurately reflect the beginning of exponential amplification and can lead to errors in quantification. Thus, relying on the standard deviation of the RFUs offers a consistent and scientifically sound method for establishing the threshold cycle in real-time