Are you new to qPCR? Have you ever wondered what this whole ‘Ct’ thing means? Why is it important? And why is early Ct’s better?
To answer these questions, we need to first understand what happens in a qPCR experiment. qPCR stands for quantitative Polymerase Chain Reaction. In a qPCR, DNA amplification is measured in real time by the fluorescent dye signals.
Let’s take a look at our lab book.
The increase in dye signal is in direct proportion to the number of PCR products. By collecting this fluorescent signal – during the exponential phase of the reaction – we are able to obtain quantitative information on the starting amount of the DNA target.
Here’s how. Let’s examine this representative qPCR amplification plot. A threshold line is drawn through the plot in the exponential phase of the curve. Reagents are plentiful during this phase, and thus there is an exact doubling of the target. From this intersection, we can now drop down to the x-axis and assign a Ct value to this amplification reaction.
Okay, so now we’ve got a Ct value, but what does it mean? And why is smaller better?
The Ct value, or threshold cycle, is the cycle number at which the fluorescent signal of the reaction crosses the threshold. The Ct value is inversely related to the starting amount of our target DNA. For example, if I have two samples, one with 200 ng of cDNA and the other with 25 ng of cDNA, I will get an earlier, or smaller, Ct value with the first one. This is because it will take fewer PCR cycles for the fluorescent signal to rise above background for the first sample. The second sample, since it is starting from less template (only 1/8 as much!) will take more cycles to reach the same level of detectable fluorescence.
So here’s the take home message: as the amount of DNA template decreases, the Ct value will increase. If the Ct value gets too high, say a Ct of 35 in a 96 well plate, it can be difficult to distinguish real signal from background. For this reason, many researchers will employ a Ct cutoff – and not use any data above that Ct value.
Researchers who want to optimize their systems will often look to the raw Ct values to compare whether a reagent results in better performance or not. Let’s say for example, you ran the same samples and assay with 2 different master mixes. A mix that results in earlier Ct’s is therefore beneficial because you will not need to use as much sample for the same level of detection. This can be especially important when dealing with limited or precious samples.
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