Loading presentation...

Present Remotely

Send the link below via email or IM

Copy

Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.

DeleteCancel

The MIQE Guidelines

No description
by

on 31 January 2016

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of The MIQE Guidelines

Conclusion
Any questions?

Acknowledgement

Thanks for your attention

Data analysis

qPCR validation

qPCR protocol

qPCR target information

Reverse transcription

Nucleic acid extraction

Sample

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

Summary

Shortcoming:
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