Selection of Reference Genes for Quantitative PCR: Identifying Reference Genes for Airway Epithelial Cells Exposed to Pseudomonas aeruginosa.

Selection of Reference Genes for Quantitative PCR: Identifying Reference Genes for Airway Epithelial Cells Exposed to Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol. 2020 Jun 10;: Authors: Hampton TH, Koeppen K, Bashor L, Stanton BA Abstract Most q-PCR experiments report differential expression relative to the expression of one or more reference genes. Therefore, when experimental conditions alter reference gene expression, q-PCR results may be compromised. Little is known about the magnitude of this problem in practice. We found that reference gene responses are common, hard to predict, and that their stability should be demonstrated in each experiment. Our reanalysis of 15 airway epithelia microarray data sets retrieved from NCBI identified no common reference gene that was reliable in all 15 studies. Reanalysis of published RNA-seq data in which human bronchial epithelial cells 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 human bronchial epithelial cells (HBEC) exposed to Pseudomonas aeruginosa (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 of airway ep...
Source: American Journal of Physiology. Lung Cellular and Molecular Physiology - Category: Cytology Authors: Tags: Am J Physiol Lung Cell Mol Physiol Source Type: research