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.
Publication types
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Research Support, Non-U.S. Gov't
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Validation Study
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B
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ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
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Caco-2 Cells
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Claudin-4
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Claudins / genetics
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DNA Primers / standards*
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Food*
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Fruit*
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Gene Expression / genetics
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Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
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Humans
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Mitochondrial Proteins / genetics
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NAD(P)H Dehydrogenase (Quinone) / genetics
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Nitric Oxide Synthase Type II / genetics
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RNA Splicing Factors
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Real-Time Polymerase Chain Reaction / methods
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Real-Time Polymerase Chain Reaction / standards*
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Reference Standards
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Ribonucleoprotein, U2 Small Nuclear / genetics
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Ribosomal Proteins / genetics
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Vegetables*
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beta 2-Microglobulin / genetics
Substances
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ABCB1 protein, human
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ATP Binding Cassette Transporter, Subfamily B
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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CLDN4 protein, human
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Claudin-4
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Claudins
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DNA Primers
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MRPL19 protein, human
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Mitochondrial Proteins
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RNA Splicing Factors
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Ribonucleoprotein, U2 Small Nuclear
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Ribosomal Proteins
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SF3A1 protein, human
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beta 2-Microglobulin
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ribosomal protein P0
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NOS2 protein, human
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Nitric Oxide Synthase Type II
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Glyceraldehyde-3-Phosphate Dehydrogenases
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NAD(P)H Dehydrogenase (Quinone)
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NQO1 protein, human