Quantitative Polymerase Chain Reaction (qPCR) is a laboratory technique used to detect and measure the amount of specific genetic material, such as DNA or RNA, within a sample. The core measurement derived from this process is the Cycle Threshold, or Ct value, which provides a numerical estimate of the initial quantity of the target sequence. Understanding the Ct value is necessary for interpreting the results of any qPCR test.
Understanding Quantitative PCR
qPCR works by copying a specific segment of genetic material repeatedly, a process known as amplification. The reaction is run in a specialized machine, a thermal cycler, that precisely controls temperature over multiple cycles. Each cycle involves steps that separate double-stranded DNA, allow primers to bind to the target, and then extend those primers to create new copies.
To monitor this process in real-time, the reaction includes a fluorescent molecule, such as a dye or a specialized probe. As new copies of the target DNA are generated, the fluorescent signal increases proportionally. In an optimized reaction, the amount of target material approximately doubles with every cycle, leading to exponential growth. The thermal cycler measures this light intensity at the end of each cycle, tracking the amplification curve.
Defining the Cycle Threshold Value
The Cycle Threshold (Ct) value is the specific cycle number at which the fluorescent signal generated by the accumulating genetic material crosses a pre-determined limit. This limit, known as the threshold line, is set just above the level of background noise inherent in the reaction. The background signal is the low-level fluorescence detected in the initial cycles before the target material has sufficiently accumulated.
The Ct value marks the point where the quantity of the newly synthesized material can be reliably distinguished from this noise. For example, if the signal crosses the threshold at cycle 18, the Ct value is 18. This numerical result is the primary output of a qPCR run and is used to quantify the starting amount of genetic material in the original sample.
Interpreting High and Low Ct Numbers
The Ct value has an inverse relationship with the initial concentration of the target material in the sample. A low Ct number indicates the fluorescent signal crossed the threshold quickly, meaning a high starting amount of the target sequence was present. Conversely, a high Ct number signifies the reaction required many cycles to generate enough copies for detection, pointing to a low starting concentration.
In diagnostic testing, the Ct value is often used as a rough indicator of the viral load. A Ct value below 20 suggests a very high concentration of genetic material, while a Ct value above 30 indicates a much lower concentration. Because amplification is exponential, a difference of just one cycle threshold represents a two-fold difference in the starting quantity. For instance, a sample with a Ct of 25 had approximately half the amount of target material compared to a sample with a Ct of 24.
Factors That Influence Ct Results
Although the Ct value measures initial concentration, several technical variables unrelated to the target quantity can influence the final number. The quality of the biological sample is a significant factor, as poorly collected or improperly stored samples may contain degraded genetic material or reaction inhibitors. These inhibitors can slow down the amplification process, artificially increasing the Ct value.
The overall efficiency of the reaction depends on the quality of the reagents used, including the primers and the enzyme. If the primers are not well-designed or the enzyme is not working optimally, the doubling of the target sequence may not occur with every cycle. This delay in detection results in a higher Ct value. Variations in the instrument’s calibration or the consistency of the threshold setting across different runs can also introduce shifts in the reported Ct numbers.