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Absolute vs. Relative Quantitation

When calculating the results of your real-time PCR (qPCR) experiment, you can use either absolute or relative quantitation.

Absolute vs. Relative Quantitation at a Glance
  Absolute Quantitation Relative Quantiation
Overview
In absolute quantitation, you quantitate unknowns based on a known quantity. First you create a standard curve; then you compare unknowns to the standard curve and extrapolate a value. In relative quantitation, you analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
Example Correlating viral copy number with a disease state. Measuring gene expression in response to a drug.

In this example, you would compare the level of gene expression of a particular gene of interest in a chemically treated sample relative to the level of gene expression in an untreated sample.


Absolute Quantitation Using the Standard Curve Method
The standard curve method for absolute quantitation is similar to the standard curve method for relative quantitation, except the absolute quantities of the standards must first be known by some independent means.

Figure 1:
Amplification Plot and Standard Curve for Absolute Quantitation
Amplification Plot and Standard Curve for Absolute Quantitation

Critical Guidelines
The guidelines below are critical for proper use of the standard curve method for absolute quantitation:

  • It is important that the DNA or RNA be a single, pure species. For example, plasmid DNA prepared from E. coli often is contaminated with RNA, which increases the A260 measurement and inflates the copy number determined for the plasmid.
  • Accurate pipetting is required because the standards must be diluted over several orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A260 value. This concentrated DNA or RNA must then be diluted 106–1012 -fold to be at a concentration similar to the target in biological samples.
  • The stability of the diluted standards must be considered, especially for RNA. Divide diluted standards into small aliquots, store at –80 °C, and thaw only once before use.

It is generally not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step.

Standards
The absolute quantities of the standards must first be known by some independent means. Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute standards. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA.


Relative Quantation

Calculation Methods for Relative Quantitation
Relative quantitation can be performed with data from all real-time PCR instruments. The calculation methods used for relative quantitation are:

  • Standard curve method
  • Comparative CT method
Figure 2:
Relative Quantiation
Relative Quantiation

 

Which Method Should I Use?

  Standard Curve Method Comparative CT Method
Overview It is easy to prepare standard curves for relative quantitation because quantity is expressed relative to some basis sample (called a calibrator), such as an untreated control.

For all experimental samples, you determine target quantity from the standard curve and divide by the target quantity of the calibrator.

Thus, the calibrator becomes the 1× sample, and all other quantities are expressed as an n-fold difference relative to the calibrator.		 This method compares the Ct value of one target gene to another (using the formula: 2ΔΔCT)—for example, an internal control or reference gene (e.g., housekeeping gene)—in a single sample.
Advantages Running the target and endogenous control amplifications in separate tubes and using the standard curve method of analysis requires the least amount of optimization and validation.
  • You don't need a standard curve, which can increase throughput because wells no longer need to be used for the standard curve samples. This also eliminates dilution errors made in creating the standard curve samples.
  • You can amplify the target and endogenous control in the same tube, increasing throughput and reducing pipetting errors.
  • When RNA is the template, performing amplification in the same tube provides some normalization against variables such as RNA integrity and reverse transcription efficiencies.
Experimental Validation See Advantages above.
  • You have to run a validation experiment to show that the efficiencies of the target and endogenous control amplifications are approximately equal.
  • To amplify the target and endogenous control in the same tube, limiting primer concentrations must be identified and shown not to affect CT values.
Critical Guidelines
  • It is important that stock RNA or DNA be accurately diluted, but the units used to express this dilution are irrelevant. If two-fold dilutions of a total RNA preparation from a control cell line are used to construct a standard curve, the units could be the dilution values 1, 0.5, 0.25, 0.125, and so on. By using the same stock RNA or DNA to prepare standard curves for multiple plates, the relative quantities determined can be compared across the plates.
  • You can use a DNA standard curve for relative quantitation of RNA. Doing this requires the assumption that the reverse transcription efficiency of the target is the same in all samples, but the exact value of this efficiency need not be known.
  • For quantitation normalized to an endogenous control, standard curves are prepared for both the target and the endogenous reference. For each experimental sample, the amount of target and endogenous reference is determined from the appropriate standard curve. Then, the target amount is divided by the endogenous reference amount to obtain a normalized target value. Again, one of the experimental samples is the calibrator, or 1× sample. Each of the normalized target values is divided by the calibrator normalized target value to generate the relative expression levels.
 For the comparative CT method to be valid, the efficiency of the target amplification (your gene of interest) and the efficiency of the reference amplification (your endogenous control) must be approximately equal.
Endogenous Control Amplification of an endogenous control may be performed to standardize the amount of sample RNA or DNA added to a reaction. For the quantitation of gene expression, researchers have used ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA (rRNA), or other RNAs as an endogenous control.  
Standards Because the sample quantity is divided by the calibrator quantity, the unit from the standard curve drops out. Thus, all that is required of the standards is that their relative dilutions be known. For relative quantitation, this means any stock RNA or DNA containing the appropriate target can be used to prepare standards.  


 

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