In a qPCR Amplification plot, where is the threshold set?

The threshold in a qPCR amplification plot is set above the background signal and within the exponential phase of the amplification curve. This is done to ensure that the threshold exceeds the noise levels that may occur at the starting phase of the reaction while capturing the true exponential growth of the PCR products.

Setting the threshold in this manner allows for accurate quantification of the target DNA. During the exponential phase, the amount of DNA doubles with each cycle, making it the most reliable point for quantification, as the increase in signal is proportional to the starting template concentration. Thus, placing the threshold above the background ensures that only the specific amplification signal, which indicates the presence of the target, is measured, avoiding potential interference from non-specific signals or fluctuations in baseline readings.

A threshold set below background would not effectively differentiate between the signal generated from the amplification and the noise inherent in the dataset, leading to unreliable results. Similarly, placing it at the peak of the curve would not account for the exponential nature of the reaction, and setting it at the baseline would not capture the amplification signal at all. Hence, the correct choice emphasizes the importance of establishing the threshold in a range that supports precise quantification of the amplifying target.