title: Normalization genes for quantitative RT-PCR in differentiated Caco-2 cells used for food exposure studies.
abstract: Exposure of food products to small-intestinal-like Caco-2 cells, combined with a gene expression based response analysis can be a valuable tool to classify potential bioactive effects of food homogenates. In order to study changes in gene expression upon food exposure, a robust set of stably expressed genes is required for normalization. Here we present a set of reference genes suitable for RT-qPCR that has been validated for exposure studies with the intestinal-like Caco-2 cell line. This study identified ribosomal phosphoprotein P0 (RPLP0) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as best reference genes. The set can be extended with β-2-microglobulin (B2M), splicing factor 3A, subunit 1 (SF3A1), and mitochondrial ribosomal protein L19 (MRPL19). Food homogenates did provoke responses in the Caco-2 cells, as was demonstrated by changed expression of NAD(P)H Quinone dehydrogenase 1 (NQO1), Claudin 4 (CLDN4), Nitric Oxide Synthase 2 (NOS2), and ATP-binding cassette, subfamily B, member 1 (ABCB1) in the same experiment. Results indicate that: i) natural food homogenates can exert effects in Caco-2 cells, and ii) stability in expression of the reference genes is not due to a lack of response of the Caco-2 cells.
authors.affiliation: Wageningen University and Research centre, Food & Biobased Research, P.O. Box 17, 6700 AA, Wageningen, The Netherlands. robert.vreeburg@wur.nl
authors.forename: Robert A M
authors.initials: RA
authors.lastname: Vreeburg
authors.forename: Shanna
authors.initials: S
authors.lastname: Bastiaan-Net
authors.forename: Jurriaan J
authors.initials: JJ
authors.lastname: Mes
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D018938:Caco-2 Cells; D062506:Claudin-4; D057167:Claudins / Q000235:genetics; D017931:DNA Primers / Q000592:standards*; D005502:Food; D005638:Fruit; D015870:Gene Expression / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D000072260:RNA Splicing Factors; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D014675:Vegetables; D001613:beta 2-Microglobulin / Q000235:genetics
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C568157:CLDN4 protein, human; D062506:Claudin-4; D057167:Claudins; D017931:DNA Primers; C496590:MRPL19 protein, human; D024101:Mitochondrial Proteins; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; D012269:Ribosomal Proteins; C088050:SF3A1 protein, human; D001613:beta 2-Microglobulin; C054675:ribosomal protein P0; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human