MIQE qPCR Guidelines, Three Years Later

MIQE qPCR Guidelines, Three Years Later

05/01/2013

Lauren Arcuri Ware

Three years ago, a group of researchers began campaigning for the adoption of guidelines that promised to improve the reproducibility of RT-qPCR data. Lauren Arcuri Ware reports on the adoption and evolution of these guidelines.

http://www.biotechniques.com/news/MIQE-qPCR-Guidelines-Three-Years-Later/biotechniques-342718.html 

PCR: living life amplified and standardized

PCR: living life amplified and standardized

by Vivien Marx
With strategies for reproducibility and quality control, scientists seek to cultivate better practices in
quantitative PCR experiments.

http://www.nature.com/nmeth/journal/v10/n5/pdf/nmeth.2443.pdf

qPCR Newsletter -- April 2013 issue -- focus on MIQE guidelines

Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is:

Update - new papers around the MIQE guidelines -  MIQE.gene-quantification.info

Update - new MIQE_qPCR APP version 1.4 for iOS available - new functions - new applications - new MIQE literature database

Update - new eBooks and Methods issues available - qPCRbooks.gene-quantification.info

GenEx - a powerful tool for qPCR data analysis - download a free trial version - GenEx.Gene-Quantification.info

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization

Background

RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts.

Results

Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions.

Conclusion

We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes.

RefGenes is available http://www.refgenes.org/rg/

and within Genevestigator at http://www.genevestigator.com

EMBO Practical Course - Single-Cell Gene Expression Analysis

EMBO Practical Course - Single-Cell Gene Expression Analysis
EMBL Heidelberg, Germany
Saturday 29 June - Friday 5 July 2013
Application deadline: Thursday 11 April 2013
http://www.embl.de/training/events/2013/SIC13-01/index.html

The analysis of transcripts in minute samples is often used to increase the knowledge in areas such as cancer development, plant physiology, neurodegenerative disorders of developmental biology. A very precise sampling crucial is crucial for the success of the approach. Depending on the nature of the sample and the goal of the experiment, there are different ways of sampling the probes. This course will present flow cytometry and laser capture microdissection as methods to obtain single cells for the subsequent gene expression analysis. The need for quantitative single-cell mRNA analysis is given by the inability of conventional RNA methods, to differentiate mRNA abundance of such limited samples. Another crucial step is data analysis, which constitutes another strong component of the course.

This course targets scientists who are entering the field of single cell analysis and want to learn about different preparation methods and gene expression profiling. The participants will learn about the complete workflow and gain understanding about the possibilities and limitations of the approach. The course will cover lectures and hands-on sessions by experts in the fields and is aimed for PhD students, postdoctoral fellows or other scientists with a solid qPCR and molecular biology background.