Real-time PCR Data Markup Language

MIQE: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

The RDML file format is a recommended element in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., Clinical Chemistry, 2009).

The aim of MIQE, coordinated by a group of research-active scientists and coordinated under the umbrella of MIBBI (Minimum Information for Biological and Biomedical Investigations) is to provide authors, reviewers and editors specifications for the minimum information that must be reported for a qPCR 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 qPCR.

icon   Download the MIQE paper

icon   Download the MIQE checklist

icon   Download the MIQE checklist

icon   Letter to Editors inviting them to adopt the MIQE standard for their papers

Checklist for Quantitative PCR Assays

Item to checkImportance
Experimental design
Definition of experimental and control groupsE
Number within each groupE
Assay carried out by core lab or investigator's lab?D
Acknowledgement of authors' contributionsD
Volume/mass of sample processedD
Microdissection or macrodissectionE
Processing procedureE
If frozen - how and how quickly?E
If fixed - with what, how quickly?E
Sample storage conditions and duration (especially for FFPE samples)E
Nucleic acid extraction
Procedure and/or instrumentationE
Name of kit and details of any modificationsE
Source of additional reagents usedD
Details of DNase or RNase treatmentE
Contamination assessment (DNA or RNA)E
Nucleic acid quantificationE
Instrument and methodE
Purity (A260/A280)D
RNA integrity method/instrumentE
RIN/RQI or Cq of 3' and 5' transcriptsE
Electrophoresis tracesD
Inhibition testing (Cq dilutions, spike or other)E
Reverse transcription
Complete reaction conditionsE
Amount of RNA and reaction volumeE
Priming oligonucleotide (if using GSP) and concentrationE
Reverse transcriptase and concentrationE
Temperature and timeE
Manufacturer of reagents and catalogue numbersD
Cqs with and without RTD*
Storage conditions of cDNAD
qPCR target information
If multiplex, efficiency and LOD of each assayE
Sequence accession numberE
Location of ampliconD
Amplicon lengthE
In silico specificity screen (BLAST, etc)E
Pseudogenes, retropseudogenes or other homologs?D
Sequence alignmentD
Secondary structure analysis of ampliconD
Location of each primer by exon or intron (if applicable)E
What splice variants are targeted?E
qPCR oligonucleotides
Primer sequencesE
RTPrimerDB identification numberD
Probe sequencesD**
Location and identity of any modificationsE
Manufacturer of oligonucleotidesD
Purification methodD
qPCR protocol
Complete reaction conditionsE
Reaction volume and amount of cDNA/DNAE
Primer, (probe), Mg++ and dNTP concentrationsE
Polymerase identity and concentrationE
Buffer/kit identity and manufacturerE
Exact chemical constitution of the bufferD
Additives (SYBR Green I, DMSO, etc.)E
Manufacturer of plates/tubes and catalog numberD
Complete thermocycling parametersE
Reaction setup (manual/robotic)D
Manufacturer of qPCR instrumentE
qPCR validation
Evidence of optimisation (from gradients)D
Specificity (gel, sequence, melt, or digest)E
For SYBR Green I, Cq of the NCTE
Standard curves with slope and y-interceptE
PCR efficiency calculated from slopeE
Confidence interval for PCR efficiency or standard errorD
r2 of standard curveE
Linear dynamic rangeE
Cq variation at lower limitE
Confidence intervals throughout rangeD
Evidence for limit of detectionE
If multiplex, efficiency and LOD of each assayE
Data analysis
qPCR analysis program (source, version)E
Cq method determinationE
Outlier identification and dispositionE
Results of NTCsE
Justification of number and choice of reference genesE
Description of normalisation methodE
Number and concordance of biological replicatesD
Number and stage (RT or qPCR) of technical replicatesE
Repeatability (intra-assay variation)E
Reproducibility (inter-assay variation, %CV)D
Power analysisD
Statistical methods for result significanceE
Software (source, version)E
Cq or raw data submission using RDMLD

MIQE checklist for authors, reviewers and editors. All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if available. If using primers obtained from RTPrimerDB, information on qPCR target, oligonucleotides, protocols and validation is available from that source.

*: Assessing the absence of DNA using a no RT assay is essential when first extracting RNA. Once the sample has been validated as RDNA-free, inclusion of a no-RT control is desirable, but no longer essential.

**: Disclosure of the probe sequence is highly desirable and strongly encouraged. However, since not all commercial pre-designed assay vendors provide this information, it cannot be an essential requirement. Use of such assays is advised against.