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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: Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

Authors
item Liu, Zonglin
item Palmquist, Debra
item Ma, Menggen - NM STATE UNIV, LASCRUCES
item Liu, Jiangbo - BRADLEY UNIV, PEORIA, IL
item Alexander, Nancy

Submitted to: Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 9, 2009
Publication Date: July 6, 2009
Citation: Liu, Z., Palmquist, D.E., Ma, M., Liu, J., Alexander, N.J. 2009. Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR. Journal of Biotechnology. 143:10-16.

Interpretive Summary: Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been realized to be the assay of choice among available techniques for messenger ribonucleic acid (mRNA) quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as an internal reference for data normalization and process since the technology appeared. However, all housekeeping genes vary under environmental stimuli, and there is no commonly accepted housekeeping gene reference available. Accurate data acquisition and data reproducibility remain challenging, and it is difficult to compare results from different experimental sets. We developed a robust standard system using a fixed threshold based on a sole exogenous transcript and a master equation derived from the universal ribonucleic acid control as a constant reference for simplified and efficient absolute mRNA quantification. This development impacts a larger research and development community for simple and accurate quantification measurement of gene expression.

Technical Abstract: The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and analysis since the technology appeared. However, all housekeeping genes vary under environmental conditions and there is no commonly accepted housekeeping gene reference available. Accurate data acquisition and data reproducibility remain challenging, and it is difficult to compare results from different experimental sources. Using yeast and a Fusarium fungus, we demonstrate the independent performance of a sole reference gene, CAB, as a constant reference of manual threshold for data acquisition, normalization, and analysis under varied conditions. A master equation with highly fitted linear relationship was obtained for reactions in varied RNA background of Saccharomyces cerevisiae and Fusarium sporotrichioides. A valid range of amplification efficiency between 95 to 100% was observed for the controls applied on the ABI real time PCR 7500 system. This newly developed robust quality control system provides a reliable reference for absolute quantification of mRNA using the qRT-PCR assay, simplifies the conventional qRT-PCR procedures, and increases data reliability, reproducibility, and throughput of the assay. It is expected that such a robust control system will significantly benefit applications for many other species.

Last Modified: 7/24/2014
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