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Why are there inconsistencies between my raw data (R) and corrected data (ΔR)?

To ensure proper data analysis by the AriaMX software, only wells containing a qPCR reaction should have dye filters activated. When running multiple assay types on a single plate, targets must be assigned to their respective reference dye.

Having fluorescence detection turned on in empty wells (Figure 1) can affect the calculations which the software makes to obtain the corrected data, ∆R log (Figure 2).  In Figure 1 all wells are defined as unknown with FAM and HEX activated.  Figure 2 shows ∆R for FAM in a sample which does not match the raw data, R, in Figure 3.  Figure 4 shows an acceptable plate setup with targets assigned in the properties window on the right side of the screen.  Figure 5 shows the proper trace for FAM in the ∆R and there is no Cq value.

Figure 1: Improper plate setup (empty wells activated and no targets assigned).

Figure 2: ∆R Log of improper plate setup.

Figure 3: Raw data of the sample in which FAM (blue) does not show growth in fluorescence because it is not influenced by the fluorescence of other wells.

Figure 4: Acceptable plate setup with only wells containing a qPCR reaction activated and targets assigned to each dye.

Figure 5: ∆R Log (acceptable plate setup).