MIQE: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
Proposed Recommendation, December 1st, 2008.

The aim of MIQE, created by Stephen Bustin 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

Download the MIQE checklist

Introduction

Quantitative polymerase chain reaction (qPCR) assays measure the copies of a specific DNA target in a sample as that sample is repeatedly passed through the polymerase chain reaction. Special qPCR machines are required to quantify the amplification products at each step of the cycle. MIQE specifies the minimum information needed for a correct interpretation of the experiment.

Checklist for Quantitative PCR Assays

1. Sample
   • Fresh - How rapidly processed?
   • Frozen - How frozen?
   • Whole vs. microdissected
   • Sample storage conditions and duration
   • Fixed - How fixed, how old?


2. Nucleic acid
   • Quantification
   • Quality/integrity
   • Inhibition dilution or spike
   • DNA contamination assessment of RNA sample
   • DNase treatment
   • Manufacturer of reagents used
   • Amount of sample used for extraction


3. Reverse treanscriptions
   • cDNA priming method + concentration
   • Amount of RNA used per reaction
   • Enzyme type and concentration
   • Detailed reaction conditions
   • Manufacturer of reagents used
   • Reaction volume
   • Storage of cDNA


4. Target
   • Database name and target gene accession number
   • Intronless, targeting of all splice variants/splice variant-specific targeting
   • Official gene symbol
   • Location of amplicon with respect to reference sequence
   • Information about (retro)pseudogenes


5. Primers and probes
   • Primer sequences
   • Location of modification
   • End concentration of primers and optional probe(s) used
   • Primer purification method
   • Manufacturer of oligonucleotides
   • Probe sequence


6. Assay details
   • Amplicon length
   • Specific BLAST or equivalent in silico specific screen
   • Experimental validation of specificity
   • NTC; Sensitivity
   • PCR efficiency, PCR efficiency standard curve slope and r-squared value
   • RTPrimerDB ID
   • Secondary structure analysis around priming sites


7. PCR Cycling
   • Amount of cDNA/DNA used per reaction
   • Detailed reaction conditions, thermocycling parameters
   • Manufacturer of reagents used
   • Manual/robotic dispensing of reagents
   • Manufacturer of plates/tubes
   • Manufacturer of real-time instrument


8. Data analysis
   • Cq value determination method
   • Treatment of NTCs and technical replicates
   • Normalisation method
   • Is r-squared value of regression curve satisfactory?
   • Has assay sensitivity been adequately evaluated and described?
   • Has assay specificity been adequately described?
   • Is the dynamic range of the assay acceptable?
   • Is the coefficient of variation for inter and intra-assay reproducibility reasonable?
   • Concordance of biological replicates
   • Analysis program
   • Assay carried out by core lab or investigator's lab
   • Acknowledgement of author's contribution to analysis and interpretation
   • Submission of Cq values of raw data using RDML

RDML guidelines (formaly known as MIqPCR)
Working draft, 4th April, 2008.

It is crucial that data acquisition, analysis and reporting become more transparent to allow reinterpretation and to guarantee compliance with quality standards. Therefore, following the example of the microarray community and their MIAME (Minimum Information About a Microarray Experiment) guidelines, we propose guidelines specifying the minimal information about qPCR experiments. A RDML guidelines compliant RDML file should contain all measured data as well as information about the samples and targets being analyzed.

In addition, data must be linked to samples and targets in an unequivocal way. Due to the complexity and diversity of experiments in which qPCR is utilized, the scope of the RDML guidelines is limited to the technology itself, which means that these guidelines can easily be integrated into other minimum information guidelines that focus on the wider experimental context. To coordinate this effort, the RDML consortium recently joined the MIBBI project (Minimum Information for Biological and Biomedical Investigations). The minimum information guidelines have been kept minimal to facilitate the creation of a compliant RDML files that make the least demand on researchers’ time, while requiring sufficient information for other researchers to interpret and reanalyze the data contained within an RDML guidelines compliant RDML file.

All information needed for the MIQE checklist can be stored in specialy designed elements or description strings inside an RDML file.

Reporting requirement for Quantitative PCR Assays

1. Administrative information
   1. Experiment description
      • Experiment description
      • Responsible person and contact details


2. Sample annotation
   1. Sample description
      • Sample ID
      • Sample description
      • cDNA synthesis method and DNAse treatment (cDNA samples only)
      • Template quantity (standard and optical calibrator samples only)
   2. Sample role in qPCR assay
      • Sample type
      • Inter run calibrator (true or false)
      • Calibrator sample (true or false)


3. Target annotation
   1. Target description
      • Target ID
      • Sequence of primers OR commercial assay description
   2. Target role in qPCR assay
      • Target type


4. Thermal Cycling Conditions Information
   1. PCR program
      • Complete description of the cycling conditions


5. Run data
   1. Instrument information
      • Plate format
      • Instrument description
      • Software description and version
   2. Information required for each well
      • Well ID
      • Sample ID
      • Target ID
      • Amplification curve fluorescence values for each data point
      • Melting curve fluorescence values for each data point
      • Quantification Cycle


6. Software requirements
   1. RDML-Support
      • Software solutions, including databases, must support the import and export of RDML files.
      • qPCR machines must allow the export of raw data for the amplification as well as for melting curves.

More detailed information about the terms used in the RDML guidelines can be found here
Download a document about the RDML guidelines.