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Reference gene selection and validation for HT29 and VK2/E6E7 human epithelial cell lines treated with probiotic and pathogenic bacteria: HT29 and EK2/V6V7 reference gene selection
Örebro University, School of Science and Technology.
2013 (English)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
Abstract [en]

The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has already been demonstrated. Investigation of the extent of this interaction is important to understanding how bacteria can be used as probiotics in the future. Currently, real-time quantitative polymerase chain reaction (qPCR) is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation must be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Although selection of an endogenous control gene is the preferred method of normalisation, this selection is often made without proper validation of the gene’s appropriateness for the study in question. In this study we used qPCR data and applied four different algorithms (genormPLUS, BestKeeper, Normfinder, and comparative ΔCq) to evaluate eight different genes as to their suitability as endogenous controls for use in studies involving HT29 (colonic) and VK2/E6E7 (vaginal) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 (PGK1) to be most appropriate for HT29 cells, and transmembrane protein 222 (TMEM222) to be the best choice for VK2/E6E7 cells. In both cell lines reference stability would be improved by use of multiple endogenous controls. This study provides recommendations for stable endogenous control genes for use in further studies involving HT29 and VK2/E6E7 cells after bacterial challenge.

Place, publisher, year, edition, pages
2013. , p. 31
Keywords [en]
polymerase chain reaction, housekeeping genes, Lactobacillus
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:oru:diva-28940ISRN: ORU-NAT/BIO-GK-2013/0002--SEOAI: oai:DiVA.org:oru-28940DiVA, id: diva2:619885
Subject / course
Biology
Presentation
2013-05-03, B219, 10:15 (English)
Uppsok
Life Earth Science
Supervisors
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Available from: 2013-05-07 Created: 2013-05-05 Last updated: 2017-10-17Bibliographically approved

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CiteExportLink to record
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