Understanding PCR Ct Vales

The PCR Ct (cycle threshold) value refers to the number of cycles needed to replicate enough DNA/RNA to be detected (crosses a threshold line). A Ct value of 20 means it took fewer cycles to produce enough DNA/RNA than a Ct of 30. The lower Ct value means there was more DNA/RNA in the sample to begin with.

Large amount of DNA/RNA in sample = Fewer cycles needed for detection = Low/Hot Ct

Smaller amounts of DNA/RNA in sample = More cycles needed for detection = Higher/Cooler Ct

Looking at the plot above, the red line on the right has a Ct ~30 (crosses the blue threshold line), the middle line has a Ct ~27 and the furthest left green line has a Ct ~25 according to the figure. (Don’t worry, the program does calculate exact Cts, which we use for reporting.) In this case, the sample associated with the red line had the least amount of DNA/RNA. Whereas the sample associated with the line furthest to the left, had the most DNA/RNA. When a Ct is recorded as 0, no DNA/RNA of the target was detected.

Johne’s example: An animal with a Ct less than or equal to 29 is considered a high Johne’s shedder, because fewer cycles were needed to detect the DNA. This correlates with a large number of bacteria. An animal with a Ct of 36- 37 took more replication cycles, meaning there is less DNA. This correlates with fewer bacteria in the sample and therefore is considered a low shedder.

Revised 10-2022

Printable version available here.