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 qPCR1 | Importance2 |
Experimental Design | |
Definition of experimental and control groups | Essential |
Number within each group | Essential |
Assay carried out by core lab or investigator's lab? | Desirable |
Acknowledgement of authors' contributions | Desirable |
Sample | |
Description | Essential |
Volume/mass of sample processed | Desirable |
Microdissection or macrodissection | Essential |
Processing procedure | Essential |
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 instrumentation | Essential |
Name of kit and details of any modifications | Essential |
Source of additional reagents used | Desirable |
Details of DNase or RNase treatment | Essential |
Contamination assessment (DNA or RNA) | Essential |
Nucleic acid quantification | Essential |
Instrument and method | Essential |
Purity (A260/A280) | Desirable |
Yield | Desirable |
RNA integrity method/instrument | Essential |
RIN/RQI or Cq of 3' and 5' transcripts | Essential |
Electrophoresis traces | Desirable |
Inhibition testing (Cq dilutions, spike or other) | Essential |
Reverse Transcription | |
Complete reaction conditions | Essential |
Amount of RNA and reaction volume | Essential |
Priming oligonucleotide (if using GSP) and concentration | Essential |
Reverse transcriptase and concentration | Essential |
Temperature and time | Essential |
Manufacturer of reagents and catalogue numbers | Desirable |
CqS with and without gene-specific primingRT | Desirable3 |
Storage conditions of cDNA | Desirable |
qPCR Target Information | |
If multiplex, efficiency and LOD of each assay | Essential |
Sequence accession number | Essential |
Location of amplicon | Desirable |
Amplicon length | Essential |
in silico specificity screen (BLAST, etc) | Essential |
Pseudogenes, retropseudogenes or other homologs? | Desirable |
Sequence alignment | Desirable |
Secondary structure analysis of amplicon | Desirable |
Location of each primer by exon or intron (if applicable) | Essential |
What splice variants are targeted? | Essential |
qPCR Oligonucleotides | |
Primer sequences | Essential |
RTPrimerDB Identification Number | Desirable |
Probe sequences | Desirable4 |
Location and identity of any modifications | Essential |
Manufacturer of oligonucleotides | Desirable |
Purification method | Desirable |
qPCR Protocol | |
Complete reaction conditions | Essential |
Reaction volume and amount of cDNA/DNA | Essential |
Primer, (probe), Mg++ and dNTP concentrations | Essential |
Polymerase identity and concentration | Essential |
Buffer/kit identity and manufacturer | Essential |
Exact chemical constitution of the buffer | Desirable |
Additives (SYBR Green I, DMSO, etc.) | Essential |
Manufacturer of plates/tubes and catalog number | Desirable |
Complete thermocycling parameters | Essential |
Reaction setup (manual/robotic) | Desirable |
Manufacturer of qPCR instrument | Essential |
qPCR Validation | |
Evidence of optimization (from gradients) | Desirable |
Specificity (gel, sequence, melt, or digest) | Essential |
For SYBR Green I, Cq of the NTC | Essential |
Standard curves with slope and y-intercept | Essential |
PCR efficiency calculated from slope | Essential |
Confidence interval for PCR efficiency or standard error | Desirable |
R2 of standard curve | Essential |
Linear dynamic range | Essential |
Cq variation at lower limit | Essential |
Confidence intervals throughout range | Desirable |
Evidence for limit of detection | Essential |
If multiplex, efficiency and LOD of each assay | Essential |
Data Analysis | |
qPCR analysis program (source, version) | Essential |
Cq method determination | Essential |
Outlier identification and disposition | Essential |
Results of NTCs | Essential |
Justification of number and choice of reference genes | Essential |
Description of normalization method | Essential |
Number and concordance of biological replicates | Desirable |
Number and stage (RT or qPCR) of technical replicates | Essential |
Repeatability (intra-assay variation) | Essential |
Reproducibility (inter-assay variation, %CV) | Desirable |
Power analysis | Desirable |
Statistical methods for result significance | Essential |
Software (source, version) | Essential |
Cq or raw data submission using RDML | Desirable |
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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