How to Export Raw Fluorescence Data from QuantStudio Real-Time PCR Systems

To export raw fluorescence data from a QuantStudio system, open your experiment in QuantStudio Design & Analysis Software (or Thermo Fisher Connect), go to Export (under the gear/settings icon or File menu), check the boxes for Raw Data and Multicomponent Data, select .xlsx or .txt as your output format, and hit Export. The resulting file will contain cycle-by-cycle fluorescence values for every well and every dye channel — which is what you actually need if you want to do anything beyond accepting the software's auto-thresholded Ct values.

That said, the specifics differ depending on which QuantStudio model you're using, which software version you're running, and whether you're working in the desktop app or the cloud platform. The rest of this post walks through the details so you don't end up with a file full of baseline-corrected, normalized ΔRn values when you wanted the raw, unprocessed fluorescence reads.

Desktop Software vs. Thermo Fisher Connect (Cloud)

Most QuantStudio users are running QuantStudio Design & Analysis Software v2.x on a connected PC. If your lab has a QuantStudio 3 or 5, you might also be accessing runs through Thermo Fisher Connect (the cloud platform). The export workflow differs enough between the two that it's worth covering separately.

Desktop (Design & Analysis Software v2.x):

1. Open your .eds experiment file.
2. Click the Export button (top toolbar, looks like a page with an arrow).
3. In the Export dialog, you'll see a list of data categories. The ones you care about are:
   • Raw Data — this gives you the raw fluorescence intensity (Rn) for each cycle, each well, each dye. This is the unprocessed signal from the detector.
   • Multicomponent Data — this breaks out the contribution of each dye after spectral unmixing (relevant for multiplex TaqMan assays or when you want to see the ROX reference signal separately).
   • Results — this is the standard output with Ct values, baselines, thresholds. You probably already have this.
4. Choose your file format. .xlsx is easiest to work with for most people. .txt (tab-delimited) is better if you're feeding it into R, Python, or another analysis tool.
5. Select an output folder and export.

Thermo Fisher Connect (Cloud):

1. Open your experiment in the browser interface.
2. Navigate to Analysis > Export.
3. The cloud platform exports a .xlsx file by default. The raw data tab is included if you check the appropriate boxes, but the labeling is slightly different — look for Raw Fluorescence Data or Multicomponent.
4. One annoyance: the cloud export sometimes omits the raw data tabs if the run was uploaded from a .eds file rather than run natively on a cloud-connected instrument. If your export is missing the raw data, download the .eds file and open it in the desktop software instead.

Older QuantStudio Real-Time PCR Software (v1.x) and runs from the QuantStudio 6 Flex or QuantStudio 7 Flex systems use essentially the same workflow, but the UI layout is a bit different — the export function is under File > Export rather than a toolbar button. The data categories are the same.

Understanding What's in the Export: Raw vs. Baseline-Corrected vs. ΔRn

This is where people get tripped up. QuantStudio software can export several flavors of fluorescence data, and they are not the same thing:

• Raw fluorescence (Rn): The actual fluorescence intensity detected in each well at each cycle, normalized to the passive reference dye (ROX or mustard, depending on your plate and master mix). This is what the detector measured. If you're using PowerUp SYBR Green Master Mix or TaqMan Fast Advanced Master Mix, ROX normalization is applied to correct for well-to-well volume differences.

• Baseline-corrected (ΔRn): Rn minus the baseline fluorescence (typically calculated from cycles 3-15, though QuantStudio's auto-baseline algorithm adjusts this per well). This is what gets plotted in the amplification plot by default. ΔRn = Rn − baseline.

• Multicomponent data: The raw fluorescence broken out by individual dye contribution after the software applies spectral calibration (the "component" data from your most recent dye calibration plate run). This is essential for troubleshooting multiplex assays because it lets you see whether spectral bleed-through is contaminating a channel.

If your goal is independent analysis — reprocessing Ct calls, fitting your own baseline, or feeding data into an external tool — you want the Raw Data (Rn) export. The ΔRn values have the software's baseline subtraction already baked in, and you can't undo that without the raw values.

If your goal is checking for spectral issues in a multiplex TaqMan setup (e.g., FAM bleed into VIC, or ROX signal dropping), you want Multicomponent Data.

File Structure and How to Work With It

The exported .xlsx file typically contains multiple sheets. The Raw Data sheet will have columns roughly like this:

For a 96-well plate with 40 cycles and one dye channel, that's 3,840 rows. A 384-well plate with two dye channels gives you 30,720 rows. The files get large — not unmanageable, but large enough that Excel starts to feel sluggish if you're trying to plot everything.

A few practical tips for handling these files:

• Pivot tables in Excel work well for reshaping the data if you want one column per well. But honestly, if you're doing this regularly, write a short script. In R, readxl::read_excel() handles the multi-sheet .xlsx exports cleanly. In Python, openpyxl or pandas.read_excel() with sheet_name='Raw Data' does the job.

• Watch the cycle numbering. QuantStudio starts at cycle 1, but some analysis methods (like LinRegPCR) expect the data to be zero-indexed or to exclude the first few cycles. Check your downstream tool's expectations.

• ROX normalization is already applied in the Rn column. If you truly want the un-normalized, absolute fluorescence values per channel, you need the Multicomponent Data export, which gives you the signal from each dye independently before normalization. This matters for some edge cases — for example, if you suspect your ROX signal is inconsistent (common with older seals or plates that have been stored improperly).

Common Export Issues and How to Fix Them

"I don't see the Raw Data option in the export dialog." This usually means you're running an older version of the Design & Analysis software (pre-2.3) or the run file was created on a different instrument model and imported. Update the software, or try re-opening the .eds file with a current version. If you're on Thermo Fisher Connect, download the .eds and use the desktop app.

"My export file has ΔRn but not Rn." You probably only checked the "Results" export box, not "Raw Data." Go back and explicitly check Raw Data. ΔRn is included in both the Results and Raw Data exports, but Rn is only in the Raw Data export.

"The data looks weird — fluorescence values are negative." Negative ΔRn values in early cycles are normal (baseline subtraction can produce small negatives). Negative Rn values are not normal and suggest a problem with the ROX normalization — likely a missing or failed spectral calibration for ROX. Re-run your dye calibration plate.

"I need to export data from many runs at once." The desktop software exports one .eds file at a time. There's no batch export in the GUI. If you have 50 runs to process, you have two options: (1) write a script using the Thermo Fisher Connect API (they have a REST API with export endpoints, though documentation is sparse), or (2) automate the desktop software with something like AutoHotKey or PyAutoGUI, though this is fragile. Alternatively, most labs just export as they go and keep a consistent folder structure.

"My Ct values don't match when I reanalyze the raw data." This is expected. QuantStudio's auto-threshold and auto-baseline algorithms are proprietary — if you re-threshold manually or use a different baseline correction method, your Ct values will shift. Differences of 0.2-0.5 Ct are common and not a sign of error. This is actually a good reason to export the raw data and standardize your own analysis pipeline across experiments.

When Raw Fluorescence Export Actually Matters

For most routine ΔΔCt experiments — three biological reps, two technical reps, one reference gene, two conditions — you don't need the raw fluorescence. The QuantStudio software's auto-analyzed Ct values are fine. Export the Results table, check that your replicate CVs are under 0.5 Ct, and move on.

Raw fluorescence export matters when you're doing something more demanding: comparing efficiency across primer sets (you need the amplification curves to fit in LinRegPCR or run Pfaffl (2001) corrections), troubleshooting an assay where the amplification plot shape looks off, validating a new primer pair, or building a standard curve and want to verify linearity directly from the curves. It also matters if you're publishing in a journal that expects deposited raw data — MIQE guidelines (Bustin et al., 2009) explicitly recommend reporting this.

If you're routinely exporting QuantStudio data for ΔΔCt analysis and want to skip the manual threshold-checking and replicate-flagging steps, VoilaPCR reads QuantStudio export files directly — upload the .xlsx and it handles baseline validation, outlier detection, and fold-change calculations with the statistical tests already applied.