Relative Quantification Algorithms Available in the AriaMx Software

Answer

This Information Applies To: Agilent AriaMx real-time PCR system, AriaMx Real-Time PCR software

Issue

The AriaMx software offers three different algorithms to calculate differences in relative expression.

Resolution

The three algorithms are as follows:

ΔΔCq (Livak) - The ΔΔCq method, also called the Livak method, relies on two assumptions. The first assumption is that the normalizer and target-of-interest have amplification efficiencies close to 100% and that the efficiencies of the targets do not differ by more than 5%. The second assumption is that the amplification efficiency of a target is consistent from one run to the next. Any run-to-run variance is not included in the calculations. The ΔΔCq method is often referred to as an approximation method and requires a validation step to confirm that efficiencies of your normalizer and target-of-interest are similar. This method is only available if the wells selected on the Analysis Criteria screen include a normalizer target.

Tip: The normalizer is a target that is known to be unaffected by the experimental treatment under investigation, and thus is found in equal quantities across all template samples. Data from the normalizer target is used to normalize the fluorescence signal of the targets of interest. It is possible to set a dye as the normalizer in Comparative Quantitation and User Defined experiments,

ΔCq - The ΔCq method is similar to the ΔΔCq method in that it relies on the same mathematical assumptions about efficiencies and consistency. Unlike the ΔΔCq method, however, no normalizer is used. Therefore the software just assumes that for every one cycle difference in the Cq value between an unknown and the calibrator, there is a two-fold difference in amplicon copy number. Since that method does not use a normalizer, there is no correction factor for sample-to-sample differences in concentration due to factors that are not directly related to biology. For example variation in the cell number that the DNA or RNA was isolated from or nucleic acid degradation from sample handling.

Pfaffl - With the Pfaffl method, the program considers the amplification efficiencies of the normalizer and target-of-interest when calculating the relative quantities¹. This method is a good choice for Comparative Quantitation experiments in which the normalizer and target-of-interest differ in amplification efficiency. The Pfaffl method does not, however, incorporate run-to-run variations in amplification efficiency. This method is only available if the wells selected on the Analysis Criteria screen include a normalizer target.

• Pfaffl, M. W. 2001 Nucleic Acids Res. 29(9),e45. DOI: 10.1093/nar/29.9.e45

Selecting the Algorithm in the AriaMx software

To select an algorithm for the relative quantity calculations:

1. Select the Relative Quantity chart on the Graphical Displays screen (Figure. 1).                                                                              

          
   
   Figure 1. Graphical Displays screen
   
   

2. In the right panel, click the downward arrowhead above the result table to display the advanced analysis parameter settings (Figure. 2).                                                                            
   

   
   
   Figure 2. Relative Quantity menu
   
   

3. Next to Mode, select the method you want to use (Figure. 3).                          
   

   
   
   Figure 3. Quantification Mode
   
   

   Learn how to effectively operate your Agilent AriaMx: AriaMx Real-Time PCR System Setup and User Guide on Agilent.com Aria Real-Time PCR Software User Manual on Agilent.com

For Research Use Only. Not for use in diagnostic procedures.

PR7001-1357