Terminology

qPCR Real-Time PCR

A quantitative method of PCR that cycles DNA until a set threshold is reached for detection of your DNA product.

• The threshold line is the point or level of detection at which a reaction reaches a fluorescent intensity above background levels.

C_q Values Quantification Cycle

The threshold line is the point or level of detection at which a reaction reaches a fluorescent intensity above background levels. Before you conduct PCR, the software in your cycler will set a threshold level.

• This is literally a line in your graph that represents a level above background fluorescence, that also intersects your reaction curve somewhere at the beginning of its exponential phase.

The Cq value is the PCR cycle number at which your sample’s reaction curve intersects this threshold line. This value tells how many cycles it took to detect a real signal from your samples.

Cq values are inverse to the amount of target nucleic acid that is in your sample, and correlate to the number of target copies in your sample.

• Lower Cq values (typically below 28 cycles) indicate high amounts of the target sequence.

• Higher Cq values (above 38 cycles) mean lower amounts of your target nucleic acid.
  • However, high Cq values may also indicate some problems with the target or the PCR set-up.

C_t Values Threshold Cycle

There is no difference between C_t and C_q value, though C_q is preferred.

Threshold Line

Generally, the fluorescence threshold is set within the exponential phase of PCR amplification and corresponds to 10 times the standard deviation of fluorescence signals during the initial 3–15 cycles of PCR.

For consistency and comparability of experimental data, it is recommended to use the same threshold setting within the same experiment and maintain consistent settings across different batches whenever possible.

Methodology

Analysis of Relative Gene Expression Data using Real-Time Quantitative PCR Livak et al. 2002 Methodology

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