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Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells

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Abstract

The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs—geNorm and Normfinder—were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.

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Acknowledgments

The authors are grateful to Nadine Aguilera for help in animal handling. This study was supported by the Institut National pour le Cancer (grant INCA-2009-175), and the Ligue contre le Cancer Department du Rhone. FMC is supported by IARC and ARC.

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Authors and Affiliations

  1. Laboratoire de Génétique du Développement, Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires (IBMM), rue des Profs. Jeener et Brachet 12, 6041, Gosselies, Belgium

    Maria Sirakov

  2. Molecular Biology Core Service, Stazione Zoologica Anton Dohrn, Naples, Italy

    Marco Borra

  3. Centre de Génétique et de Physiologie Moléculaire et Cellulaire, Université Claude Bernard Lyon 1, Villeurbanne, France

    Francesca Maria Cambuli & Michelina Plateroti

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  1. Maria Sirakov
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  2. Marco Borra
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  3. Francesca Maria Cambuli
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  4. Michelina Plateroti
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Correspondence to Maria Sirakov.

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Maria Sirakov and Marco Borra contributed equally to this study.

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Sirakov, M., Borra, M., Cambuli, F.M. et al. Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells. Mol Biotechnol 54, 930–938 (2013). https://doi.org/10.1007/s12033-012-9643-3

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