MIQE - Minimum Information for Publication of Quantitative Real-Time PCR Experiments
An international consortium has published in 2009 the MIQE guidelines to help researchers produce more reliable data. The MIQE guidelines describe the minimum Information that must be provided for publication of Quantitative real-time PCR Experiments. These guidelines should enable other investigators to reproduce the experiments and increase consistency between laboratories. These guidelines will also help reviewers to assess the validity of the protocols used. A list of the MIQE guidelines is presented below :
|
Checklist of the MIQE guidelines |
Importance |
|
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 - how and how quickly? |
Essential |
|
If fixed - with what, 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 RT |
Desirable |
|
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 (or context of the sequence) |
Desirable |
|
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 |
For more information regarding the MIQE guidelines, click here.
Our experiments meet the recommendations of the MIQE guidelines and all relevant information will be provided to our users together with their data. For any question regarding our procedures, click here to contact us.