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Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium

Author

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  • Jose V Die
  • Ransom L Baldwin
  • Lisa J Rowland
  • Robert Li
  • Sunghee Oh
  • Congjun Li
  • Erin E Connor
  • Maria-Jose Ranilla

Abstract

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Suggested Citation

  • Jose V Die & Ransom L Baldwin & Lisa J Rowland & Robert Li & Sunghee Oh & Congjun Li & Erin E Connor & Maria-Jose Ranilla, 2017. "Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium," PLOS ONE, Public Library of Science, vol. 12(2), pages 1-13, February.
    • Handle: RePEc:plo:pone00:0172674
    DOI: 10.1371/journal.pone.0172674
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    References listed on IDEAS

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    1. Michael Lawrence & Wolfgang Huber & Hervé Pagès & Patrick Aboyoun & Marc Carlson & Robert Gentleman & Martin T Morgan & Vincent J Carey, 2013. "Software for Computing and Annotating Genomic Ranges," PLOS Computational Biology, Public Library of Science, vol. 9(8), pages 1-10, August.
    2. Wuzheng Zhu & Yaqiu Lin & Honghai Liao & Yong Wang, 2015. "Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle," PLOS ONE, Public Library of Science, vol. 10(3), pages 1-15, March.
    3. Francis Jacob & Rea Guertler & Stephanie Naim & Sheri Nixdorf & André Fedier & Neville F Hacker & Viola Heinzelmann-Schwarz, 2013. "Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines," PLOS ONE, Public Library of Science, vol. 8(3), pages 1-8, March.
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