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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: Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls

Authors
item LIU, ZONGLIN
item SLININGER, PATRICIA

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: August 30, 2007
Publication Date: August 30, 2007
Citation: Liu, Z., Slininger, P.J. 2007. Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls [abstract]. American Society for Microbiology.

Technical Abstract: Real time quantitative RT-PCR is considered as standard for gene expression and mRNA estimate. As a calibration standard, a conserved control gene such as housekeeping genes is commonly used for data normalization and analysis. A significant problem has been observed with increased applications; however, that many so- called housekeeping genes are not consistently performed as a control reference under different conditions. Recently, we developed universal external RNA controls that can be applied to both microarray and qRT-PCR and allow comparison of expression data from different platforms. This set of universal RNA controls is a robust standard and has been applied to yeast and other fungi for absolute mRNA quantification successfully. However, limited information is available for bacterial applications. In this study, we investigate its utilization for Pseudomonas fluorescens Pf-5, a plant disease biocontrol agent, in response to different culture conditions for desiccation tolerance studies. Four control genes from cattle and soybean were used. Primers and probes were designed for both SYBR Green and TaqMan probes based chemistry. The universal controls performed consistently with a highly fitted linear regression (R2=0.99) for cycle numbers as a function of log (mRNA) over conditions independent form culture conditions, strain sources, and different cell treatments. We demonstrate that this universal control provides reliable and consistent reference for absolute mRNA quantification of P. fluorescens Pf-5.

Last Modified: 8/19/2014
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