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HomeQuantitative PCR (qPCR)MIQE – Minimum Information for Publication of Quantitative Real-Time PCR Experiments

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

The potential applications for Quantitative Real-Time PCR (qPCR) have increased exponentially since the first description (Higuchi, 1993). However, researchers have been frustrated by complications such as contamination, insufficient amplification, low sensitivity, and uncertainty about what constitutes a suitable statistical analysis. Until recently, there has been a lack of consensus about how to deal with these obstacles.

An international research team, including Dr. Tania Nolan, our Global Manager for Applications and Technical Support, published The MIQE (pronounced Mykee) Guidelines in 2009 to address the challenges of performing dependable qPCR measurements. By following MIQE, you are certain to produce more reliable data and will:

  • Promote experimental transparency
  • Ensure consistency between laboratories
  • Maintain the integrity of the scientific literature

Our qPCR services, including primer and probe designs, assay protocol development, troubleshooting, and data analysis support, adhere to MIQE, which allows you to publish or bring your product to market faster and with confidence.

The MIQE Checklist Quick Reference Guide

The MIQE Checklist for qPCR1Importance2
Experimental Design
Definition of experimental and control groupsEssential
Number within each groupEssential
Assay carried out by core lab or investigator's lab?Desirable
Acknowledgement of authors' contributionsDesirable
Sample
DescriptionEssential
Volume/mass of sample processedDesirable
Microdissection or macrodissectionEssential
Processing procedureEssential
If frozen - method and how quickly?Essential
If fixed - with what and how quickly?Essential
Sample storage conditions and duration (especially for FFPE samples)Essential
Nucleic Acid Extraction
Procedure and/or instrumentationEssential
Name of kit and details of any modificationsEssential
Source of additional reagents usedDesirable
Details of DNase or RNase treatmentEssential
Contamination assessment (DNA or RNA)Essential
Nucleic acid quantificationEssential
Instrument and methodEssential
Purity (A260/A280)Desirable
YieldDesirable
RNA integrity method/instrumentEssential
RIN/RQI or Cq of 3' and 5' transcriptsEssential
Electrophoresis tracesDesirable
Inhibition testing (Cq dilutions, spike or other)Essential
Reverse Transcription
Complete reaction conditionsEssential
Amount of RNA and reaction volumeEssential
Priming oligonucleotide (if using GSP) and concentrationEssential
Reverse transcriptase and concentrationEssential
Temperature and timeEssential
Manufacturer of reagents and catalogue numbersDesirable
CqS with and without gene-specific primingRTDesirable3
Storage conditions of cDNADesirable
qPCR Target Information
If multiplex, efficiency and LOD of each assayEssential
Sequence accession numberEssential
Location of ampliconDesirable
Amplicon lengthEssential
in silico specificity screen (BLAST, etc)Essential
Pseudogenes, retropseudogenes or other homologs?Desirable
Sequence alignmentDesirable
Secondary structure analysis of ampliconDesirable
Location of each primer by exon or intron (if applicable)Essential
What splice variants are targeted?Essential
qPCR Oligonucleotides
Primer sequencesEssential
RTPrimerDB Identification NumberDesirable
Probe sequencesDesirable4
Location and identity of any modificationsEssential
Manufacturer of oligonucleotidesDesirable
Purification methodDesirable
qPCR Protocol
Complete reaction conditionsEssential
Reaction volume and amount of cDNA/DNAEssential
Primer, (probe), Mg++ and dNTP concentrationsEssential
Polymerase identity and concentrationEssential
Buffer/kit identity and manufacturerEssential
Exact chemical constitution of the bufferDesirable
Additives (SYBR Green I, DMSO, etc.)Essential
Manufacturer of plates/tubes and catalog numberDesirable
Complete thermocycling parametersEssential
Reaction setup (manual/robotic)Desirable
Manufacturer of qPCR instrumentEssential
qPCR Validation
Evidence of optimization (from gradients)Desirable
Specificity (gel, sequence, melt, or digest)Essential
For SYBR Green I, Cq of the NTCEssential
Standard curves with slope and y-interceptEssential
PCR efficiency calculated from slopeEssential
Confidence interval for PCR efficiency or standard errorDesirable
R2 of standard curveEssential
Linear dynamic rangeEssential
Cq variation at lower limitEssential
Confidence intervals throughout rangeDesirable
Evidence for limit of detectionEssential
If multiplex, efficiency and LOD of each assayEssential
Data Analysis
qPCR analysis program (source, version)Essential
Cq method determinationEssential
Outlier identification and dispositionEssential
Results of NTCsEssential
Justification of number and choice of reference genesEssential
Description of normalization methodEssential
Number and concordance of biological replicatesDesirable
Number and stage (RT or qPCR) of technical replicatesEssential
Repeatability (intra-assay variation)Essential
Reproducibility (inter-assay variation, %CV)Desirable
Power analysisDesirable
Statistical methods for result significanceEssential
Software (source, version)Essential
Cq or raw data submission using RDMLDesirable

1Clinical chemistry Copyright 2009 by AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY, INC. Reproduced with permission of AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY, INC in the format Internet posting via Copyright Clearance Center.

2All essential information must be submitted with the manuscript. Desirable information should be submitted if available. If primers are from RTPrimerDB, information on qPCR target, oligonucleotides, protocols, and validation is available from that source.

3Assessing the absence of DNA using a no-reverse transcription assay is essential when first extracting RNA. Once the sample has been validated as DNA-free, inclusion of no-reverse transcription control is desirable but no longer essential.

4Disclosure of the probe sequence is highly desirable and strongly encouraged. However, because not all commercial pre-designed assay vendors provide this information, it cannot be an essential requirement. Use of such assays is discouraged.

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