In this newsletter we will review the advantages of each method and factors to consider using each of the most popular relative quantification methods:
- Comparative CT Method (ΔΔCt)
- Relative Standard Curve Method
Comparative CT Method (ΔΔCT)
Comparative CT method (ΔΔCT) uses a reference sample and an endogenous control to determine the relative quantity of target nucleic acid sequence in sample. Standard curves are not required to run on each plate, which result in reduced reagent usage.
The Comparative CT Method uses arithmetic formulas to achieve the result for relative quantification. It is possible to eliminate the use of standard curves and to use the ΔΔCT Method for relative quantification as long as the PCR efficiencies between the target(s) and endogenous control(s) are relatively equivalent.
The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by the arithmetic formula: 2 –ΔΔCT
This method is useful when a high number of targets and/or number of samples are tested. Consider this method when using a high throughput strategy and when validating NGS results
Relative Standard Curve Method
Relative Standard Curve Method also uses a reference sample and an endogenous control to determine the relative quantity but considered to give highly accurate quantitative results because unknown sample quantitative values are interpolated from the standard curve(s).
This method requires the least amount of validation because the PCR efficiencies of the target and endogenous control do not have to be equivalent.
Relative standard curve method uses a set of relative standards from which unknown samples are quantitated. Standard curves for relative quantification are easy to prepare because quantity is expressed relative to some basis sample, such as the calibrator, a sample used as the basis for comparing results.
For relative quantification, any stock cDNA, RNA, or DNA containing the appropriate target can be used to prepare standards.
For the proper use of relative standard curves:
- Stock cDNA, RNA, or DNA must be accurately diluted, but the units used to express the dilutions are not important.
- A DNA standard curve can be used for relative quantification of RNA. This assumes 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.
Because quantification should be normalized to an endogenous control, standard curves are prepared for both the target and the endogenous reference. For each experimental sample, you determine the amount of target and endogenous reference from the appropriate standard curve. Since this method requires that each reaction plate contains standard curves for each target thus requires more reagents and more space on a reaction plate, consider this method when testing low numbers of targets and small numbers of samples and if you are looking for very discrete expression changes.For more information follow this link:
Guide to Performing Relative quantification of Gene Expression Using Real-Time Quantitative PCR.