Technical note: Validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli

S. Bougarn, P. Cunha, F. B. Gilbert, F. Meurens, P. Rainard

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-α). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data.

Original languageEnglish
Pages (from-to)2425-2430
Number of pages6
JournalJournal of Dairy Science
Volume94
Issue number5
DOIs
Publication statusPublished - 1 May 2011

    Fingerprint

Keywords

  • Innate immunity
  • Mammary epithelial cells
  • Normalization
  • QPCR
  • Reference genes

ASJC Scopus subject areas

  • Food Science
  • Animal Science and Zoology
  • Genetics

Cite this