Custom DNA Probes

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

 
Click here for The MIQE Guidelines

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, Sigma's 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

Sigma's 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

After reviewing and becoming familiar with the MIQE publication, download the checklist below and display it on any convenient laboratory or office location.

 

Download The MIQE Checklist (188 Kb PDF)

 

Mouse over any term in green in the table below for a definition of that term.

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 - 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 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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