If you are performing quantitative PCR (qPCR), it is very important to know and understand what the ct value is. In this month’s newsletter we will discuss the critical role of the ct value in qPCR, its changing nomenclature, how it is calculated and what happens when things sometimes go wrong.
All Nomenclature
We would like to emphasize that a ct value has been given many names over the years as technology has developed and changed, including the ones listed below. These values are all the same, although their meaning may be a bit strange when translated into Turkish. To standardize qPCR terminology, MIQE (Minimum Information guidelines for the Publication of Quantitative Real-Time PCR Assays) recommends the use of the Cq value.
Ct – (threshold cycle) threshold cycle
Cp – (crossing point) crossing point
TOP – (take-off point) take-off point
Cq – (quantification cycle) quantification cycle
What is this Cq Value?
Real-time PCR (qPCR) is used to measure the absolute amount of a target sequence or to compare the relative amounts of a target sequence between samples. This technique is monitored in real time by amplification of a specific fluorescent signal that binds to the target sequence. Although fluorescent probes are sequence-specific, background fluorescence can also occur during most qPCR experiments. It is crucial to be able to ignore or account for these background signals if we want to gather meaningful information about the target sequence. This problem is addressed by two factors in qPCR: Threshold line and Cq value.
Threshold is the point at which the target sequence reaches a detection-level fluorescence intensity. It is the signal that reflects a statistically significant increase, which can be calculated automatically or manually.
The Cq value is the number of PCR cycles where the reaction curve of the sample intersects the threshold line. The qPCR instrument software calculates and plots the Cq value for each sample. Cq is used to calculate the starting DNA copy number because the Cq value is inversely proportional to the starting amount of the target. At the end of each cycle your qPCR instrument will collect the fluorescence data and the fluorescence signal that you see as a straight line from the first PCR cycle until about 15 PCR cycles will start to show logarithmic amplification, rising above the threshold line after the 15th cycle.
Figure. Threshold level and Cq-value in a real-time PCR amplification curve.
Cq Factors affecting the value of
There are many factors that can influence this value.
References