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The MIQE Guidelines

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on 31 January 2016

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Transcript of The MIQE Guidelines

Any questions?


Thanks for your attention

Data analysis

qPCR validation

qPCR protocol

qPCR target information

Reverse transcription

Nucleic acid extraction


E: Essential D: Desirable

Experimental design

The goal of MIQE

Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE).

What is MIQE?

Standard Curve

Dissociation Curve

Amplification Plots

qPCR is a very powerful method for detection and quantification of mRNA transcription levels of a selected gene of interest.
Enables researchers to produce millions of copies of specific DNA sequence in one to two hours.

Quantitative Real-Time PCR (qPCR)

qPCR oligonucleotides

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT.
Clin Chem. 2009 Apr;55(4):611-22. Epub 2009 Feb 26.

Nomeclature (Terminology)

Where does poor qPCR results come from?

Highly misleading

Inefficient assay performance

“Breakthrough of the Year 2005”

The Story Behind ...

Presented by: Mehrnaz Montazeri
Supervisor: Dr Alireza Fazeli
Date: 11/03/2014
Bustin et al. 2009

PCR and The MIQE Guidelines


1. Inhibition testing (RNA Spike Control Kits).
2. RQI (RNA Quality Index) and RIN (RNA Integrity Number) using either 28S/18S or Agilent 2100 Bioanalyser.
3. Primer Secondary Structure (M-fold).

They were 85 parameters in MIQE guideline and:
We cover 80 of the parameters.

The progress so far...

After the first author left the unit, analysis of his original data revealed that data from the real-time PCR were analysed incorrectly .
Certain data points were removed and despite his colleagues attempt to re-analyse the data, they failed to reproduce the findings of the article.

Later on ...

Data Analysis

Quantification PCR

Reverse Transcription and Amplification

RNA Analysis

RNA Extraction and Purification

The qPCR approach

Highly variable results

Inefficient assay performance

Highly misleading

Quantitative Polymerase Chain Reaction (qPCR)
Full transcript