Table 1. dMIQE checklist for authors, reviewers, and editors.a
Item to checkImportanceItem to check 2Importance
Experimental designdPCR oligonucleotides
    Definition of experimental and control groups.E    Primer sequences and/or amplicon context sequence.bE
    Number within each group.E    RTPrimerDB (real-time PCR primer and probe database) identification number.D
    Assay carried out by core lab or investigator's lab?D    Probe sequences.bD
    Power analysis.D    Location and identity of any modifications.E
Sample    Manufacturer of oligonucleotides.D
    Description.E    Purification method.D
        Volume or mass of sample processed.EdPCR protocol
        Microdissection or macrodissection.E    Complete reaction conditions.E
        Processing procedure.E        Reaction volume and amount of RNA/cDNA/DNA.E
        If frozen—how and how quickly?E        Primer, (probe), Mg++ and dNTP concentrations.E
        If fixed—with what, how quickly?E        Polymerase identity and concentration.E
    Sample storage conditions and duration (especially for formalin-fixed, paraffin-embedded samples).E        Buffer/kit catalogue no. and manufacturer.E
Nucleic acid extraction        Exact chemical constitution of the buffer.D
    Quantification—instrument/method.E        Additives (SYBR green I, DMSO, etc.).E
    Storage conditions: temperature, concentration, duration, buffer.E    Plates/tubes Catalogue No and manufacturer.D
    DNA or RNA quantificationE    Complete thermocycling parameters.E
    Quality/integrity, instrument/method, e.g. RNA integrity/R quality index and trace or 3′:5′.E    Reaction setup.D
    Template structural information.E    Gravimetric or volumetric dilutions (manual/robotic).D
    Template modification (digestion, sonication, preamplification, etc.).E    Total PCR reaction volume prepared.D
    Template treatment (initial heating or chemical denaturation).E    Partition number.E
    Inhibition dilution or spike.E    Individual partition volume.E
    DNA contamination assessment of RNA sample.E    Total volume of the partitions measured (effective reaction size).E
    Details of DNase treatment where performed.E    Partition volume variance/SD.D
    Manufacturer of reagents used and catalogue numberD    Comprehensive details and appropriate use of controls.E
    Storage of nucleic acid: temperature, concentration, duration, buffer.E    Manufacturer of dPCR instrument.E
RT (If necessary)dPCR validation
    cDNA priming method + concentration.E    Optimization data for the assay.D
    One- or 2-step protocol.E    Specificity (when measuring rare mutations, pathogen sequences etc.).E
    Amount of RNA used per reaction.E    Limit of detection of calibration control.D
    Detailed reaction components and conditions.E    If multiplexing, comparison with singleplex assays.E
    RT efficiency.DData analysis
    Estimated copies measured with and without addition of RT.bD    Mean copies per partition (λ or equivalent).E
    Manufacturer of reagents used and catalogue number.D    dPCR analysis program (source, version).E
    Reaction volume (for 2-step RT reaction).D    Outlier identification and disposition.E
    Storage of cDNA: temperature, concentration, duration, buffer.D    Results of no-template controls.E
dPCR target information    Examples of positive(s) and negative experimental results as supplemental data.E
    Sequence accession number.E    Where appropriate, justification of number and choice of reference genes.E
    Amplicon location.D    Where appropriate, description of normalization method.E
        Amplicon length.E    Number and concordance of biological replicates.D
        In silico specificity screen (BLAST, etc.).E    Number and stage (RT or dPCR) of technical replicates.E
        Pseudogenes, retropseudogenes or other homologs?D    Repeatability (intraassay variation).E
            Sequence alignment.D    Reproducibility (interassay/user/lab etc. variation).D
        Secondary structure analysis of amplicon and GC content.D    Experimental variance or CI.dE
    Location of each primer by exon or intron (if applicable).E    Statistical methods used for analysis.E
    Where appropriate, which splice variants are targeted?E    Data submission using RDML (Real-time PCR Data Markup Language).D
  • a All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if possible.

  • b Disclosure of the primer and probe sequence is highly desirable and strongly encouraged. However, since not all commercial predesigned assay vendors provide this information, when it is not available assay context sequences must be submitted [Bustin et al. (48)].

  • c Assessing the absence of DNA using a no-RT assay (or where RT has been inactivated) is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of a no-RT control is desirable, but no longer essential.

  • d When single dPCR experiments are performed, the variation due to counting error alone should be calculated from the binomial (or suitable equivalent) distribution.