Selection of reference genes for quantitative PCR: identifying reference genes for airway epithelial cells exposed to Pseudomonas aeruginosa.

American Journal of Physiology. Lung Cellular and Molecular Physiology
Thomas H HamptonBruce A Stanton

Abstract

Most quantitative PCR (qPCR) experiments report differential expression relative to the expression of one or more reference genes. Therefore, when experimental conditions alter reference gene expression, qPCR results may be compromised. Little is known about the magnitude of this problem in practice. We found that reference gene responses are common and hard to predict and that their stability should be demonstrated in each experiment. Our reanalysis of 15 airway epithelia microarray data sets retrieved from the National Center for Biotechnology Information (NCBI) identified no common reference gene that was reliable in all 15 studies. Reanalysis of published RNA sequencing (RNA-seq) data in which human bronchial epithelial cells (HBEC) were exposed to Pseudomonas aeruginosa revealed that minor experimental details, including bacterial strain, may alter reference gene responses. Direct measurement of 32 TaqMan reference genes in primary cultures of HBEC exposed to P. aeruginosa (strain PA14) demonstrated that choosing an unstable reference gene could make it impossible to observe statistically significant changes in IL8 gene expression. We found that reference gene instability is a general phenomenon and not limited to studies ...Continue Reading

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Citations

Aug 8, 2021·International Journal of Molecular Sciences·Athanassios FragoulisLucy Kathleen Reiss

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Datasets Mentioned

BETA
GEOmetadb
GDS2287

Methods Mentioned

BETA
PCR
pulldown
RNA-seq

Software Mentioned

ScanGEO
GEOmetadb
TaqMan
edgeR
GeoMean
NormqPCR R package
R
Shiny

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