Normalization genes for quantitative RT-PCR in differentiated Caco-2 cells used for food exposure studies

Food Funct. 2011 Feb;2(2):124-9. doi: 10.1039/c0fo00068j. Epub 2010 Dec 13.

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

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • Caco-2 Cells
  • Claudin-4
  • Claudins / genetics
  • DNA Primers / standards*
  • Food*
  • Fruit*
  • Gene Expression / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Humans
  • Mitochondrial Proteins / genetics
  • NAD(P)H Dehydrogenase (Quinone) / genetics
  • Nitric Oxide Synthase Type II / genetics
  • RNA Splicing Factors
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Ribonucleoprotein, U2 Small Nuclear / genetics
  • Ribosomal Proteins / genetics
  • Vegetables*
  • beta 2-Microglobulin / genetics

Substances

  • ABCB1 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • CLDN4 protein, human
  • Claudin-4
  • Claudins
  • DNA Primers
  • MRPL19 protein, human
  • Mitochondrial Proteins
  • RNA Splicing Factors
  • Ribonucleoprotein, U2 Small Nuclear
  • Ribosomal Proteins
  • SF3A1 protein, human
  • beta 2-Microglobulin
  • ribosomal protein P0
  • NOS2 protein, human
  • Nitric Oxide Synthase Type II
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • NAD(P)H Dehydrogenase (Quinone)
  • NQO1 protein, human