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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS-TOLERANT MICROBES FOR LOWER COST PRODUCTION OF BIOFUELS AND BIOPRODUCTS

Location: Crop Bioprotection Research

Title: Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR

Authors
item Liu, Zonglin
item Slininger, Patricia

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 16, 2006
Publication Date: February 23, 2007
Citation: Liu, Z., Slininger, P.J. 2007. Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR. Journal of Microbiological Methods. 68:486-496.

Interpretive Summary: With rapid advances in genome sequencing, gene expression analysis using microarray and real time qRT-PCR has been widely used for biological studies. Data reliability and reproducibility of microarray and qRT-PCR experiments are significant issues for application of gene and genomic expression research. Results derived from different platforms of microarray and qRT-PCR are not always in agreement. It is difficult to assess variations between the two different platforms for quantitative expression data. Consistent and standard quality controls for different platforms are necessary for validation, confirmation, and interpretation of gene expression data. Currently, there is no such standard control available. Acquisition of high-quality gene expression measurement remains challenging. In this study, we developed a set of six universal external RNA controls applied in microbial gene expression analysis for spotted 70-mer DNA long oligo microarrays and qRT-PCR using TaqMan and Sybr Green. Using examples of a bacterium and industrial ethanologenic yeast, we demonstrated that this set of quality controls can be universally applied to different platforms of microarray and qRT-PCR for gene expression analysis for microbial applications. The universal control serves as an unbiased normalization reference for consistent data acquisition, defines a valid range of linearity, calibrates quantification dynamics, indicates detection limit of mRNA abundance, and estimates variation of the entire microarray and the qRT-PCR experiment. It makes it possible to compare data derived from different experiments and different platforms of microarray and real time qRT-PCR including different chemistry of TaqMan and SYBR Green. This research impacts the gene expression analysis and application community involved in basic research, clinical, and industrial applications.

Technical Abstract: We presented a set of universal external RNA quality controls for microbial mRNA expression analysis across different platforms of DNA oligo microarray and real time qRT-PCR, including using different chemistry of SYBR Green and TaqMan. The control is a powerful tool to guard reliability and reproducibility of gene expression data by providing unbiased normalization reference, valid ranges of linearity, quantification dynamics, data validation and confirmation, and variation estimate of gene and genomic expression experiments.

Last Modified: 4/19/2014
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