MIQE what’s that?
When writing dPCR materials and methods for a paper have you ever pondered what information you should include? This is where the MIQE guidelines will really help. Guidelines for minimum information required for publication of a digital PCR (dPCR) experiment were published by JF Huggett et al. in 2013. These were a follow-up to minimum information requirements for publication of real-time PCR experiments (qPCR) suggested by SA Bustin et al. in 2009. MIQE guidelines for qPCR and dPCR publications are divided into the following categories: essential information and desirable information. Essential Information must be included in the submitted manuscript or accompanying supplemental material. Desirable Information should be included in the submitted manuscript and is intended to help the reader understand the study.
MIQE guidelines are simple checklists intended to help researchers provide standardized information when publishing their quantitative PCR results. The goal is to help standardize nomenclature and decrease variability in the quality of published quantitative PCR data. MIQE got its name from the initial checklist published in 2009 for real-PCR experiments – Minimum Information for Publication of Quantitative real-time PCR Experiments (MIQE). A recently published update now provides a checklist for digital PCR – Minimum Information for Publication of Quantitative Digital PCR Experiments (dMIQE). MIQE checklist requirements specific to dPCR relate to reaction partitioning and data analysis.
What’s in the MIQE Guidelines?
MIQE guidelines include checklists of Essential Information and Desirable Information.
- Essential Information must be submitted as part of your manuscript (body or supplemental materials).
- Desirable Information should be submitted with your manuscript, if possible. This is information that would further help the reader understand your study.
Essential Information includes:
- Experimental design, including controls
- Sample description, microdissection or macrodissection, preparation & storage
- Nucleic acid extraction methods, quantification, quality assessment
- Target sequence accession number, in silico screening info, primer location
- Primer sequence or amplicon context sequence if not available, primer modifications
- PCR Protocol details – PCR reaction conditions, reaction volume, reagents, thermocycling parameters
Note: Requirements specific to dPCR are partition number and partition volumes
- PCR Instrument manufacturer
- Validation e.g. specificity and multiplex info if applicable
Note: additional requirements for qPCR – calibration curves, linear dynamic range, LOD info
- Data analysis program/software used, statistical methods, outliers, normalization method, NTC results, reference gene justification, experiment repeatability
Note: dPCR-specific requirements – mean copies per partition, experimental variance
Desirable Information includes:
- Experiments performed in PI’s lab or core lab?
- Manufacturer & catalog # of reagents
- Amplicon location, amplicon secondary structure analysis
- Info on pseudogenes, retropseudogenes or other homologs if applicable
- Sequence alignment
- RTPrimerDB identification number
- Probe sequence, if not proprietary
- Oligo manufacturer and purification method
- PCR buffer chemical constitution
- Consumables catalog number & manufacturer
- Reaction set-up
- Optimization data
- Experiment reproducibility
- Data submission using RDML
Digital PCR-specific desirable information:
Gravimetric or volumetric dilutions (manual/robotic)
Total PCR reaction volume prepared
Partition volume variance/SD
Limit of detection of calibration control
Need an App for That?
Yes, there is an app for that! You may already be using the initial MIQE qPCR app released in 2011. An updated 2013 release now includes digital PCR checklists and digital PCR dedicated references (http://appshopper.com/reference/miqe-qpcr).
What’s the Benefit of Following MIQE Guidelines?
MIQE guidelines help standardize quantitative PCR experimental data, making it easier for other researchers to understand and replicate your study. That means more reliable data sharing and more breakthroughs for our scientific community. An example of a publication that has integrated them is Sedlak et al (2014) Identification of Chromosomally Integrated Human Herpesvirus 6 by Droplet Digital PCR Clinical Chemistry v. 60, p.765-772.
Huggett, JM et al. (2013) The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clinical Chemistry 59: 892-902.
Bustin, SA et al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry 55: 611-622.
Image Credit: Mufidah Kassalias