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United States Department of Agriculture

Agricultural Research Service

Title: Universal External Rna Quality Controls for Mrna Expression Analysis Using Microbial DNA Oligo Microarray and Real Time Quantitative Rt-Pcr

Authors
item Liu, Zonglin
item Slininger, Patricia

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 13, 2005
Publication Date: September 6, 2005
Citation: Liu, Z., Slininger, P.J. 2005. Universal external RNA quality controls for mRNA expression analysis using microbial DNA oligo microarray and real time quantitative RT-PCR [abstract]. Eighth International Meeting of the Microarray Gene Expression Data Society. Poster #P161, p. 45-46.

Technical Abstract: With rapid advances of genomic sequence and development of biotechnology, gene expression analysis using microarray and real time quantitative RT-PCR (qRT-PCR) has been widely used in a broad range of fields in biology. Quality control of microarray and qRT-PCR experiments has become an important issue and drawn ever increasing attention since the technical application emerged. To assist quantification expression measurement, exogenous nucleic acid control is applied for microarray experiments and a consistent endogenous gene is commonly used as a standard reference for qRT-PCR applications. However, performance of control genes varies depending upon host RNA background, experimental conditions, sequence preference, and even dye effects. Acquisition of high quality gene expression measurements remains challenging. Results derived from different platforms of microarray and qRT-PCR for mRNA expression are not always in agreement. The reliability of microarray and qRT-PCR data is critical for obtaining meaningful biological insight from genomic expression analysis. Moreover, consistent and standard quality controls for different platforms are necessary for validation and interpretation of gene expression data. Currently, there is no such standard control available. In this study, we developed a set of external RNA quality controls for gene expression analysis which can be applied universally to both DNA oligo microarrays and qRT-PCR. Thus, quantitative expression measurement from different platforms of microarray and qRT-PCR, including TaqMan and Syber green I can be compared using this universal control. The mRNA transcripts applied as spiked-in universal controls demonstrated highly consistent performance in response to extreme experimental conditions, including harsh inhibitive fermentation stress conditions. Therefore, it provides 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. This presentation will demonstrate the creation of the universal controls for microarray and qRT-PCR, experimental design and application for gene expression analysis using these controls, and data comparison from the different platforms.

Last Modified: 10/30/2014
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