AAT Bioquest

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

Posted March 25, 2022


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 differentiationAbsolute QuantificationRelative Quantification
What it determinesExpression levels in absolute numbers of copiesFold changes in expression between two samples
Known/Unknown template amountsThe 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 involvesUnknowns 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 determinedThe 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.
Additional resources

Relative and Absolute Quantitative Real-Time PCR-Based Quantifications of hcnC and phlD Gene Transcripts in Natural Soil Spiked with Pseudomonas sp. Strain LBUM300

Helixyte™ Green *20X Aqueous PCR Solution*