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Short answer
A Ct (cycle threshold) value is the cycle number at which a reaction’s fluorescence curve crosses a threshold line. It is a relative measure of how much target was in the reaction: less starting template means a higher Ct. Cq is the current name for the same quantity, and a Ct is only comparable when threshold, baseline and chemistry are held consistent.
Definition and scope: Ct, Cq and cycle threshold
Thermo Fisher’s real-time PCR learning centre defines Ct as the intersection between an amplification curve and a threshold line, and describes it as a relative measure of the concentration of target in the PCR reaction. The MIQE 2.0 guidelines (Clinical Chemistry 2025;71(6):634) use Cq, the quantification cycle, “formerly known as Ct or Cp”. So “ct values in qpcr”, “cq values qpcr” and “cycle threshold qpcr” all point at one number; Cq is the term the guideline asks authors to report.
This page covers what the number means and what moves it. For converting Ct values into fold changes, see the delta-delta Ct data analysis guide; for quantifying against standards, see building and validating a qPCR standard curve.
How a Ct is read in practice
Each cycle the instrument records fluorescence. The baseline is subtracted to give a baseline-corrected signal (Thermo Fisher’s delta Rn is Rn minus baseline), and a threshold line is drawn across the amplification plots. Thermo Fisher states that the threshold must be set in the linear phase of the amplification plot. The cycle where each curve crosses that line is its Ct.
Because the signal needs more cycles to reach the threshold when there is less template, the Ct value increases with a decreasing amount of template. A sample with a Ct of 22 therefore started with more target than one at 28 run under the same conditions, but the Ct itself is a cycle number, not a concentration.
For background on the chemistry that produces the fluorescence, see the qPCR and RT-qPCR guide and the TaqMan qPCR guide.
What moves a Ct: a checklist
MIQE 2.0 states that Cq depends on several factors, including the sample processing method, fluorescence acquisition and data analysis. The analysis choices it singles out are the ones most often left unreported. Use this list to check whether two Ct values can be put side by side.
| Factor | Why it changes the Ct | Source |
|---|---|---|
| Threshold placement | Must sit in the linear phase; a different level gives a different crossing cycle. Thermo Fisher uses the same threshold level when comparing reactions. | Thermo Fisher; MIQE 2.0 |
| Fixed or per-run threshold | MIQE 2.0 asks whether thresholds are consistent across runs or determined individually for each run. | MIQE 2.0 |
| Baseline correction | The baseline must be subtracted from each reaction individually. Sloped baselines can significantly affect the shape and position of amplification curves, and manual adjustment of the early cycles defining the baseline may be required. | MIQE 2.0 |
| Reagents and run conditions | Ct values from PCR reactions run under different conditions or with different reagents cannot be compared directly. | Thermo Fisher |
| Sample processing and acquisition | Named by MIQE 2.0 as factors on which Cq depends. | MIQE 2.0 |
Reaction efficiency and the standard curve
Thermo Fisher notes that a standard-curve slope of -3.3 ± 10% reflects an efficiency of 100% ± 10%. The slope links Ct to template amount across a dilution series, which is why efficiency should be checked before Ct differences are turned into quantities. The melt curve analysis guide covers a separate check, whether the signal that produced the Ct came from the intended product.
Reporting Ct and Cq values: a MIQE 2.0 checklist
- State the method of baseline correction and Cq determination.
- Report the frequency and Cq of negative controls.
- Collect raw fluorescence data and make it available on request.
- Call an unknown positive only if its Cq precedes that of the negative control by at least 5 cycles.
- Avoid further statistical tests on Cq values themselves.
The wider reporting framework is laid out in the MIQE qPCR reporting checklist.
Common misunderstandings
Ct and Cq are different measurements
They are not. MIQE 2.0 introduced Cq as the replacement name for Ct (and Cp); the value is read the same way.
A Ct is a concentration
It is a cycle number and a relative measure of target. Converting it to an amount needs a standard curve or a comparative method with checked efficiency.
A late Ct means a negative result
For validated qualitative assays, MIQE 2.0 says a positive reaction should be reported irrespective of the Cq value, and that the LLOQ or LOD should not be used as cut-offs for reporting negative findings. It also notes that the LOD cannot be below 3 target copies, that variability increases below 10 copies per reaction, and that at 90% efficiency the LLOQ is reached at 35 cycles.
FAQ
What is the difference between Ct and Cq?
Nothing in the measurement. Cq (quantification cycle) is the term used in MIQE 2.0, formerly known as Ct or Cp.
What is the threshold in qPCR?
A fluorescence level set in the linear phase of the amplification plot. The cycle at which a curve crosses it is the Ct.
Why does Ct change between runs?
Reagents, run conditions, sample processing, fluorescence acquisition, baseline correction and threshold choice can all shift it. Thermo Fisher says Ct values from reactions run under different conditions or reagents cannot be compared directly.
Does a high Ct value mean the sample is negative?
Not by itself. In qualitative assays MIQE 2.0 says positives should be reported regardless of Cq, and LOD or LLOQ should not be used as cut-offs for negatives.
Sources
- Thermo Fisher Scientific, Real-time PCR: Understanding Ct, checked 2026-10-29.
- Bustin SA et al., MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines, Clinical Chemistry 2025;71(6):634, checked 2026-10-29.
