Donnerstag, 1. März 2012

Q&A: Bio-Rad Scientists Discuss Case Study Demonstrating MIQE Importance in qPCR Experiments | PCR Insider | PCR/Sample Prep | GenomeWeb

First published in 2009 in the journal Clinical Chemistry, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines — better known as the MIQE guidelines — were designed to provide researchers with a roadmap for improving the quality and reliability of their qPCR data.
Since that time, the molecular biology research community has slowly adopted the guidelines, and many qPCR instrument and reagent vendors have done their part to help encourage their customers to follow MIQE protocols.

There is still work to be done, however, and some vendors have taken a more active role than others in disseminating information about MIQE to their customers. To wit, Bio-Rad earlier this month published a case study on its websitedemonstrating how neglecting some of the key steps in the MIQE guidelines can lead to flawed data and erroneous conclusions.

In the study, researchers from Bio-Rad and the Jewish General Hospital at McGill University studied the effect of RNA sample quality and reference gene stability on gene expression data obtained using qPCR.

More specifically, they used the minichromosome maintenance protein MCM7 as a model target gene to investigate the importance of appropriate reference gene selection. They also varied RNA sample quality from their breast cancer samples to determine its effect on data.

Following the MIQE guidelines, they observed a significant increase in gene expression of MCM7 between normal and tumor samples when using high-quality and high-purity RNA with normalization using stable reference genes. However, they obtained inconclusive and even opposite results when using poor-quality RNA samples and unstable reference genes

Last week, PCR Insider spoke about the case study with its lead author, Sean Taylor, a field application specialist at Bio-Rad, and Francisco Bizouarn, an international field application scientist in Bio-Rad's gene expression division. Following is an edited transcript of the interview.

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