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MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) is the reporting standard journals expect a qPCR methods section to satisfy. Most desk rejections and reviewer 2 methods-section flags on qPCR papers trace back to the same handful of missing disclosures — not to bad science, but to a methods paragraph that doesn’t say what was actually done. This page turns the checklist into a worksheet: what to write down while you’re at the bench, organized the way a reviewer actually reads a methods section, section by section.
MIQE and MIQE 2.0: which version to cite
The original guidelines — Bustin SA, Benes V, Garson JA, et al., “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments,” Clinical Chemistry 2009;55(4):611–622 (doi:10.1373/clinchem.2008.112797) — introduced a checklist split into “essential” (E) and “desirable” (D) items.
MIQE 2.0, published 24 April 2025, is the current revision and the one to cite in a new methods section: Bustin SA, Ruijter JM, van den Hoff MJB, et al., “MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines,” Clinical Chemistry 2025;71(6):634–651 (doi:10.1093/clinchem/hvaf043). The structural change matters for how you use the checklist: MIQE 2.0 replaces the essential/desirable tiering with a single unified Yes/No checklist, organized into five sections — reagent preparation, sample preparation, reverse transcription, qPCR protocol, and data analysis — with footnotes marking items that are highly desirable or only conditionally applicable. A lab SOP or manuscript template that still frames MIQE as an “essential vs. desirable” split is citing the 2009 structure and is out of date.
Because the full checklist text sits behind a paywall (Clinical Chemistry/Oxford Academic), the worksheet below reflects the five-section framework and the categories of information each section requires — it is not a verbatim reproduction of the journal’s numbered table. Check the primary source directly, via your institution’s subscription, before submitting a manuscript where checklist compliance will be verified against the published table.
The reporting worksheet, by MIQE 2.0 section
Use this as a pre-submission pass: for each row, can you point to the sentence in your methods section that answers it? If not, that’s the gap a reviewer will flag.
| Section | What to disclose | What a missing entry looks like to a reviewer |
|---|---|---|
| Reagent preparation | Primer and probe sequences (or a public accession/catalog number if proprietary), amplicon length, source and lot of master mix/enzyme, storage conditions for primers/probes/template. | “Standard primers were used” with no sequence or accession — unreproducible by definition, and the single most common qPCR methods complaint in peer review. |
| Sample preparation | Sample source and type, extraction/purification method and kit, nucleic acid quantification method, and a nucleic acid integrity measure (RIN/RQI, or a 3′:5′ integrity ratio for RNA) rather than concentration alone. | A concentration (ng/µL) with no integrity metric — MIQE treats a quantity number without a quality number as incomplete, because degraded template can still yield an apparently normal Cq. |
| Reverse transcription | Priming strategy (oligo-dT, random hexamer, or gene-specific), RT enzyme and reaction conditions, and whether RT efficiency or RT-minus controls were assessed. | An RT-qPCR paper that never states the priming strategy — oligo-dT and random-hexamer priming can give different relative yields across transcripts, so this is not interchangeable detail. |
| qPCR protocol | Reaction volume and concentrations, cycling conditions, PCR efficiency and R² from a standard curve (efficiency conventionally reported as acceptable in the 90–110% range), the Cq determination method (fixed threshold vs. baseline-corrected), and no-template control (NTC) results. | A reported fold-change with no efficiency/R² figure anywhere in the paper — without it, a reviewer cannot tell whether the delta-Cq math the results depend on was even valid for that assay. |
| Data analysis | Normalization method and, if using reference genes, evidence they were validated as stable for that experimental system (not assumed); replicate structure (technical replicates per sample, biological replicates per group); statistical test and software/version used. | “Normalized to GAPDH” with no stability check — GAPDH and other common housekeeping genes are not universally stable, and MIQE treats an unvalidated reference gene as a real threat to the result, not a formality. |
What reviewers actually reject on, ranked by how often it shows up
- No efficiency/R² reported. Relative quantification (delta-delta Cq and its variants) assumes near-100% amplification efficiency; reporting a fold-change with no efficiency figure gives the reviewer no way to check that assumption held.
- No NTC (no-template control) data. A missing or unreported NTC is read as “contamination and primer-dimer amplification were never ruled out” — regardless of whether they actually were.
- Unvalidated reference gene(s). Normalizing to a single assumed-stable gene without a stability check (geNorm- or NormFinder-style analysis, or at minimum a stated rationale) is one of the most common major-revision comments on qPCR-based papers.
- Single technical replicate, or technical and biological replicates conflated. MIQE expects both replicate types reported separately — technical replicates quantify assay noise, biological replicates quantify the effect you’re actually claiming. Collapsing them overstates confidence in the result.
- Missing primer sequences or amplicon identity evidence. Either the sequences themselves, or evidence the amplicon is what it’s claimed to be (melt curve plus a gel or sequencing check for dye-based assays — see the melt curve guide linked below). A single sharp melt peak alone is necessary but not sufficient evidence of specificity.
- No RNA/DNA integrity measure. A concentration figure with nothing describing template quality, for a study drawing conclusions from low-abundance targets in particular.
- Cq/Ct terminology and threshold-setting left unstated. MIQE 2.0 uses Cq (quantification cycle) as the standard term rather than the instrument-specific Ct/Cp labels different platforms use, and expects the threshold or baseline-correction method to be stated — not just the resulting number, since threshold placement is an operator decision that changes Cq.
Technical vs. biological replicates: the distinction MIQE actually cares about
A technical replicate is the same nucleic acid sample run through the qPCR reaction (or, more strictly, through extraction onward) more than once — it estimates assay and pipetting noise. A biological replicate is an independently sampled biological unit (a separate animal, culture, patient sample, or experiment) — it estimates the variability of the effect itself. Reporting three technical replicates of one biological sample as “n=3” does not support a claim about biological variability, and MIQE 2.0’s data-analysis section expects both counts stated explicitly and not conflated.
FAQ
Is MIQE mandatory for journal submission?
It depends on the journal. Some journals (particularly in molecular biology and clinical diagnostics) require or explicitly reference MIQE compliance in their author guidelines; many others don’t name it directly but their reviewers apply its substance anyway — the items above (efficiency, NTC, replicate structure, reference-gene validation) are standard reviewer expectations for any qPCR-based result regardless of whether the journal cites MIQE by name.
What actually changed between MIQE and MIQE 2.0?
The core content is similar in substance, but MIQE 2.0 (2025) replaced the original essential/desirable two-tier checklist with a single unified Yes/No checklist across the five sections above, with footnotes for conditionally-applicable items. Cite MIQE 2.0 in new manuscripts; MIQE 2009 remains the historical reference for the essential/desirable framing if you’re discussing older literature that used it.
Do I have to validate reference genes for every single experiment?
MIQE 2.0 expects evidence the normalization gene(s) are stable for your specific experimental system — cell type, treatment, tissue, or condition — not a one-time validation reused indefinitely across unrelated experiments. A gene stable across a cell line’s growth curve is not automatically stable under a drug treatment or a disease-state comparison.
What’s the difference between Cq and Ct?
They refer to the same measurement — the cycle at which fluorescence crosses a set threshold. Ct (“cycle threshold”) and Cp (“crossing point”) are older, platform-specific terms; MIQE adopted Cq (“quantification cycle”) as the standard, instrument-agnostic term, and MIQE 2.0 continues to use it. Using Ct in a manuscript isn’t wrong, but MIQE-aligned papers increasingly use Cq, and mixing both terms in the same paper without reconciling them reads as inconsistent.
Related CASRAI guides
- qPCR melt curve analysis: reading peaks, primer dimers and the confirmatory check — the specificity-evidence half of the qPCR protocol section above, worked in detail.
- qPCR and RT-qPCR: how they differ, and how to run one properly — the underlying method this checklist reports on.
- RNA Integrity Number (RIN): how it is computed and how to read a trace — the integrity metric the sample-preparation section above requires.
- Publisher reproducibility checklists: Nature Reporting Summary and MDAR — how a MIQE-compliant methods section maps onto a journal’s own reproducibility checklist.
