Performing CNV Analysis on the AriaMx

Answer

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

Issue

A Copy Number Variation (CNV) experiment is a type of Comparative Quantitation experiment where expected differences are small. Often with fold changes of 0.5 (for one copy loss) to two (for two copy gain). Probe-based assays are more suitable since they are more specific but the assays need to be developed/tested for detecting such small differences.

Steps to Follow

If a CNV experiment is run on the AriaMx, and the plate setup is correct, the AriaMx software will automatically analyze the data.

For a CNV experiment the following must be defined (Figure 1):

• A calibrator sample (calibrator) with a known copy number for the target-of-interest, ideally copy number = 2.
• Unknown samples (unknown).
• A normalizer assay (N).
• No template control (NTC).
• Optionally, a passive reference dye (R), such as ROX, to normalize for the assay signals.
• Ideally, a sample with a known deletion (copy number = 1) as a deletion control (not shown in Figure 1) would also be included. 

Figure 1. Example of a plate setup containing a calibrator sample, unknown sample, and a no template control (NTC) 

Like other comparative quantitation experiments, CNV assays can be done with or without standard curves. If the plate setup is complete with calibrator sample and normalizer assay but without standards, the AriaMx software will automatically calculate the fold difference by applying the ΔΔCq method. The ΔΔCq method assumes 100% efficiencies for both assays (target-of-interest assay and normalizer assay).

To calculate the copy number (CN) for the unknown sample:

• For the unknown and calibrator assays, calculate the ΔCqs (the difference in Cq between the target-of-interest and normalizer).
• Then calculate the ΔΔCq  (the difference between the unknown and calibrator ΔCqs).
• Calculate the fold-change. Fold change = 2^-ΔΔCq.
• Multiply the fold change by the known CN of the calibrator (Figure 2). 

Figure 2. Procedure to calculate the copy number in the test sample

If desired, standard curves can be run for the target-of-interest and normalizer assays, or reaction efficiencies can be determined separately. The standard curve or reaction efficiencies can then be used for a fold-change calculation that considers the amplification efficiencies (Pfaffl). To switch between analysis methods, click inside the Relative Quantity graph within the Graphical Displays tab. Then select either ΔΔCq (with 100 % reaction efficiencies assumed) or Pfaffl (for empirically determined reaction efficiencies) (Figure 3). 

Figure 3. Two analysis methods available in the Aria Software. The option to select ΔΔCq or Pfaffl method of quantitation is highlighted with a red rectangle.

For an example of how comparative quantitation data looks on the AriaMx, open the demo data for comparative quantitation (multiplex) that can be found saved at this location: C:\Users\Public\Public Documents\Agilent Aria\Sample Experiments.

Learn how to effectively operate your Agilent AriaMx real-time PCR system:  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.

PR7001-1970