What is the difference between Absolute Quantification and Relative Quantification in qPCR?

Question

Accepted Answer

Absolute quantification and relative quantification are two ways that are used to calculate the results of a qPCR experiment. There are a few significant differences between the two.

Basis of differentiation
Absolute Quantification
Relative Quantification

What it determines
Expression levels in absolute numbers of copies
Fold changes in expression between two samples

Known/Unknown template amounts
The precise amount of the message or template used for the curve is known. The absolute quantification standard curve provides the final answer.
Although the template is known to contain the message of interest in high abundance, its absolute amount may or may not be known.

What the process involves
Unknowns are quantified based on a known quantity. A standard curve is first created. Then the unknowns are compared to the standard curve and a value is extrapolated.
Changes in gene expression in a given sample are analyzed relative to another reference sample such as an untreated control sample.

How the answer is determined
The standard curve provides the final answer.
The calibration curve result for the target gene is normalized to that of a housekeeping gene in the same sample. The normalized numbers are then compared between samples to obtain a fold change.

Basis of differentiation	Absolute Quantification	Relative Quantification
What it determines	Expression levels in absolute numbers of copies	Fold changes in expression between two samples
Known/Unknown template amounts	The precise amount of the message or template used for the curve is known. The absolute quantification standard curve provides the final answer.	Although the template is known to contain the message of interest in high abundance, its absolute amount may or may not be known.
What the process involves	Unknowns are quantified based on a known quantity. A standard curve is first created. Then the unknowns are compared to the standard curve and a value is extrapolated.	Changes in gene expression in a given sample are analyzed relative to another reference sample such as an untreated control sample.
How the answer is determined	The standard curve provides the final answer.	The calibration curve result for the target gene is normalized to that of a housekeeping gene in the same sample. The normalized numbers are then compared between samples to obtain a fold change.

What is the difference between Absolute Quantification and Relative Quantification in qPCR?

Posted March 25, 2022