Endpoint Real-time and Rt Assays: Three Different Jobs
PCR versus qPCR versus RT-qPCR are three nucleic-acid assays with three different claims. Endpoint PCR asks whether a product of an expected size appeared. qPCR asks how the fluorescence curve crossed a threshold during cycling. RT-qPCR adds reverse transcriptase so the input can be RNA. Adding a dye or an enzyme is not a synonym upgrade. Prefer the name that matches the claim you will write. Reject the habit of calling every tube “PCR.” The PCR control checklist still owns the lane map. Tm versus annealing still owns two numbers on a cycler. This page only separates the three assay jobs.
Adding Fluorescence or Reverse Transcriptase Changes the Claim
MIQE 2.0 is a qPCR publication standard, not a cloning-PCR manual, and it is useful here because it refuses to treat “PCR” as one claim. Real-time quantitative PCR reports a quantification cycle. Reverse-transcription qPCR is a different workflow because RNA must become cDNA before that cycle means anything. Endpoint PCR, the cloning and colony-screen workhorse, usually reports a band. Those are not three brand names for one reaction. After the job is named, ZettaNote can store which assay was run, with the control map that assay actually needs. The notebook does not turn an endpoint gel into a fold-change.
Three Assays, Three Allowed Claims
The table is a naming gate. It is not a 2026 instrument rank.
| Assay | Input the claim assumes | What the result is allowed to mean | What it is not |
|---|---|---|---|
| Endpoint PCR | DNA template | A product of an expected size was or was not visible after cycling | Not a copy-number or fold-change claim |
| qPCR | DNA template (or already-made cDNA) | Fluorescence crossed a threshold at a recorded cycle under a stated chemistry | Not proof the starting material was RNA |
| RT-qPCR | RNA, then reverse transcriptase, then qPCR | A RNA-derived cDNA signal, only if no-RT and no-template controls are interpreted | Not interchangeable with a DNA qPCR on the same target |
Prefer endpoint PCR when the job is “is the insert there” or “did the colony carry the band.” Prefer qPCR when the job is a DNA quantification or a cDNA plate that has already been reverse-transcribed elsewhere. Prefer RT-qPCR when the biological claim is about RNA abundance. Digital PCR is a fourth assay and is not defined here. Relative-quantification math and the meaning of Cq are later pages in the Wave9 plan; this page stops at the assay name.
Where People Collapse the Names
The usual collapse is calling a SYBR colony screen “qPCR” because the cycler can watch fluorescence. If you never use the curve as a quantity, you ran a real-time endpoint, not a quantification. The second collapse is calling a DNA standard curve “RT-qPCR” because the project is about expression. Without reverse transcriptase on that RNA, the claim is not about transcripts. The third collapse is treating a no-template tube as the only control an RT-qPCR needs. NTC versus other negatives owns that class split. MIQE 2.0 treats no-RT as a distinct control for RT-qPCR. Name the assay first so the control set has somewhere to attach.
Record the Assay You Actually Ran
Write the assay name, the input class, and the claim you are allowed to publish before you store the file. Relative quantification and the meaning of Cq are later jobs; they cannot repair a misnamed assay. ZettaNote can hold that sentence next to the cycler program or the gel. It is a record surface, not a qPCR instrument. If the notebook still says “PCR” for an RNA fold-change, the claim is misnamed. Do not fix the name by adding a dye in software.
Frequently Asked Questions
Is qPCR just PCR with a dye?
No. A dye or probe lets you watch accumulation in real time. The claim becomes a threshold cycle, not a band on a gel. If you only score a product at the end, say so.
Does RT-qPCR measure DNA the same way qPCR does?
No. RT-qPCR starts from RNA and requires reverse transcriptase. A DNA qPCR on the same locus is a different input and a different claim.
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