QuantStudio Design & Analysis 2: From Export to Fold Change

Design & Analysis 2 changed the QuantStudio export in a way that quietly broke a lot of people's spreadsheets and scripts: the columns are now Sample, Target and Cq, where the classic software wrote Sample Name, Target Name and CT. The data is the same. The headers are not, and anything that looked for the old names, including most lab Excel templates and until recently some analysis tools, finds nothing.

Here is the export that works from DA2, what the new columns mean, and the direct route from that file to a fold-change table with statistics.

Exporting results from Design & Analysis 2

1. Open the run (your .eds file) in Design & Analysis 2, on the desktop app or on Thermo Fisher Connect.
2. Click Analyze if the run has not been analyzed, and check the threshold and baseline on the amplification plot. DA2's auto-threshold is usually right for TaqMan assays and usually too low for SYBR assays with a high baseline. Fix it here, because the exported Cq values are whatever the threshold says.
3. Export → Results. Choose xlsx (one workbook, a Results sheet with the table and a Sample Setup and Amplification Data sheet alongside) or csv (one file per table). The xlsx is the easier one to keep track of.
4. Leave the default columns selected. The ones that matter downstream are Well Position, Sample, Target, Task, Cq, and Amp Status.

The exported Results sheet starts with a block of metadata, file name, instrument type, serial number, then the table. Software that scans for the header row handles this; a spreadsheet template that expects the header on row 1 does not, which is the second-most-common reason a DA2 file "does not work."

What the new columns mean

Cq replaces CT. Same number: the fractional cycle at which the fluorescence crossed the threshold. The MIQE guidelines prefer "Cq" (quantification cycle) because "Ct" was an Applied Biosystems term; DA2 adopted the standard.

Amp Status is new and worth reading. AMP means the curve crossed threshold with a sigmoidal shape. NO_AMP means it did not, and the Cq is reported as Undetermined. INCONCLUSIVE means the software could not decide, usually a late drifting curve, and you should look at that well before trusting it. A sample with INCONCLUSIVE on your target and AMP on the reference is the classic low-expression case: treat the Cq as a floor, not a measurement.

Cq Confidence (0 to 1) and Amp Score are DA2's curve-quality metrics. A confidence below about 0.8 on a well that otherwise looks fine usually means a noisy baseline. Neither replaces looking at replicate agreement, which is the QC that actually matters for fold change.

Task is UNKNOWN, STANDARD, or NTC, inherited from the plate setup. If every row says UNKNOWN including your water wells, the plate was set up without tasks, and your NTCs will be treated as samples until something tells the analysis otherwise. Naming the wells NTC fixes this downstream even when the task was not set.

From the export to a fold-change table

Drop the xlsx (or the Results csv) onto VoilàPCR. As of October 2026 the QuantStudio parser reads both header dialects, so a DA2 export is recognized the same way a classic one is: the Results sheet is found past the metadata block, Undetermined becomes a missing Cq rather than a number, NTC tasks become no-template controls, and STANDARD tasks with their concentrations feed the standard-curve method if you choose it.

Pick the reference gene. If you ran GAPDH or ACTB or 18S on the plate, it will be at the top of the list. Pick the control group, which is inferred from sample names when they follow the Condition_replicate pattern. Analyze.

The ΔΔCt math is Livak and Schmittgen (2001): for each sample, ΔCt is target Cq minus reference Cq; for each group, ΔΔCt is the group's mean ΔCt minus the control group's mean ΔCt; fold change is 2^−ΔΔCt. Statistics run on the ΔCt values with a Welch t-test for two groups or one-way ANOVA with Holm correction for more, because ΔCt is approximately normal and fold change is not. The figure shows the fold change with error bars from the biological replicates and significance brackets from those tests.

Two DA2-specific things to watch

Multiplex runs. If you multiplexed target and reference in one well (FAM and VIC), DA2 exports two rows per well, one per target, which is exactly what the analysis needs. Do not de-duplicate on well position in Excel first; that throws away one of the two targets.

Omitted wells. The Omit column is TRUE for wells you excluded inside DA2. They are still in the export. Most tools, including VoilàPCR, honor the flag, but if you are feeding the file to your own script, filter on it, or your excluded outlier comes right back.

If you would rather not export at all, VoilàPCR also reads the .eds file directly, which is a separate post. Either way, the first analysis on your own data runs without an account.