Real-time PCR Data Markup Language

dMIQE: Minimum Information for Publication of Quantitative Digital PCR Experiments

The RDML file format is a recommended element in the minimum information for publication of quantitative digital PCR experiments (dMIQE) guidelines (Huggett et al., Clinical Chemistry, 2020)..

The aim of dMIQE, coordinated by a group of research-active scientists, is to provide authors, reviewers and editors specifications for the minimum information that must be reported for a digital PCR experiment in order to ensure its relevance, accuracy, correct interpretation and repeatability. A checklist, which should be submitted along with the paper, is available for authors in preparing a manuscript employing digital PCR.

icon   Download the dMIQE paper

icon   Download the dMIQE checklist

Checklist for digital qPCR Assays

ITEM TO CHECKPROVIDED        COMMENT
Y/N
1. SPECIMEN
Detailed description of specimen type and numbers
Sampling procedure (including time to storage)
Sample aliquotation, storage conditions and duration
2. NUCLEIC ACID EXTRACTION
Description of extraction method including amount of sample processed
Volume of solvent used to elute/resuspend extract
Number of extraction replicates
Extraction blanks included?
3. NUCLEIC ACID ASSESSMENT AND STORAGE
Method to evaluate quality of nucleic acids
Method to evaluate quantity of nucleic acids (including molecular weight and calculations when using mass)
Storage conditions: temperature, concentration, duration, buffer, aliquots
Clear description of dilution steps used to prepare working DNA solution
4. NUCLEIC ACID MODIFICATION
Template modification (digestion, sonication, pre-amplification, bisulphite etc.)
Details of repurification following modification if performed
5. REVERSE TRANSCRIPTION
cDNA priming method and concentration
One or two step protocol (include reaction details for two step)
Amount of RNA added per reaction
Detailed reaction components and conditions
Estimated copies measured with and without addition of RT*
Manufacturer of reagents used with catalogue and lot numbers
Storage of cDNA: temperature, concentration, duration, buffer and aliquots
6. dPCR OLIGONUCLEOTIDES DESIGN AND TARGET INFORMATION
Sequence accession number or official gene symbol
Method (software) used for design and in silico verification
Location of amplicon
Amplicon length
Primer and probe sequences (or amplicon context sequence)**
Location and identity of any modifications
Manufacturer of oligonucleotides
7. dPCR PROTOCOL
Manufacturer of dPCR instrument and instrument model
Buffer/kit manufacturer with catalogue and lot number
Primer and probe concentration
Pre-reaction volume and composition (incl. amount of template and if restriction enzyme added)
Template treatment (initial heating or chemical denaturation)
Polymerase identity and concentration, Mg++ and dNTP concentrations***
Complete thermocycling parameters
8. ASSAY VALIDATION
Details of optimisation performed
Analytical specificity (vs. related sequences) and limit of blank (LOB)
Analytical sensitivity/LoD and how this was evaluated
Testing for inhibitors (from biological matrix/extraction)
9. DATA ANALYSIS
Description of dPCR experimental design
Comprehensive details negative and positive of controls (whether applied for QC or for estimation of error)
Partition classification method (thresholding)
Examples of positive and negative experimental results (including fluorescence plots in supplemental material)
Description of technical replication
Repeatability (intra-experiment variation)
Reproducibility (inter-experiment/user/lab etc. variation )
Number of partitions measured (average and standard deviation )
Partition volume
Copies per partition (λ or equivalent ) (average and standard deviation)
dPCR analysis program (source, version)
Description of normalisation method
Statistical methods used for analysis
Data transparency


dMIQE2020 checklist for authors, reviewers and editors. Authors should fill detail whether information is provided. Where 'yes' is selected use comment box to detail location of information or to include the information. Where 'no' is selected use comment box to outline rationale for omission. Sections 4 and 5 may not apply depending on experiment.

*: 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.

**: Disclosure of the primer and probe sequence is highly desirable and strongly encouraged. However, since not all commercial pre-designed assay vendors provide this information when it is not available assay context sequences must be submitted (Bustin et al. Primer sequence disclosure: A clarification of the miqe guidelines. Clin Chem 2011;57:919-21.)

***: Details of reaction components is highly desirable, however not always possible for commercial disclosure reasons. Inclusion of catalogue number is essential where component reagent details are not available.