normalization data transformation protocol
Microarray data was processed using limma package in R 2.15.1. Loess within array normalisation and Aquantile between array normalisation was applied, without background correction. ID_REF = VALUE = Normalized log2 ratio (Cy5/Cy3)
array scanning protocol
The slides were scanned using a GenePix Professional 4200A scanner (Molecular Devices, Sunnyvale, CA, USA). Spot identification and quantification was performed with GenePix 6.0 software (Molecular Devices). The images were also manually checked and any poor quality spots were removed before statistical analysis.
The labelled samples were hybridized onto Agilent Technologies 44k whole human genome oligonucleotide arrays (G4112A) in a loop design with dye-swap using the Agilent gene expression hybridisation kit (5188-5242-A). RNA (750 ng of each sample per array) was mixed with the control target, fragmented for 30 min at 60°C in the dark and hybridised onto arrays in a rotation oven at 60°C, at 4 rpm for 17 hrs.
The RNA samples were labelled using the Agilent Low RNA Input Linear Amplification Kit PLUS (5183-3523). The total RNA (500 ng) was amplified and reverse transcribed to cDNA using T7-polymerase. The cDNA was labelled with either cyanine3 or cyaninine5 labelled CTP dye (Perkin-Elmer/NEN Life Sciences, Boston, MA, USA).
nucleic acid extraction protocol
Total RNA was extracted from the Caco-2 cells using TRIzol (Invitrogen, Auckland, New Zealand) according to the manufacturer’s instructions. Extracted RNA was purified using RNeasy mini columns (QIAGEN, San Diego, CA, USA) and RNA quality was assessed using an RNA 6000 NanoLabChip kit with the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). All RNA samples had RIN > 8.
sample treatment protocol
After 18 days the media was removed from the Caco-2 monolayers and replaced with one of 1) cell culture media (M199 in 1% non-essential amino acids); 2), AGR1487 (OD 600 nm of 0.9) suspended in cell culture media; and 3), AGR1485 (OD 600 nm of 0.9) suspended in cell culture media. After 8 hours of exposure the treatment solutions were removed and the monolayers were rinsed with PBS prior to RNA extraction.
Caco-2 cells were cultured in Medium 199 (M199; Invitrogen) containing 10% (v/v) fetal bovine serum (GIBCO), 1% (v/v) nonessential amino acids (MEM nonessential amino acids 100x solution; Sigma-Aldrich) and 1% Penicillin-Streptomycin (10,000 U penicillin G sodium salt and 10,000 mg streptomycin sulphate in 0.85% (v/v) saline; GIBCO) at 37°C in 5% CO₂. The media was replaced every 3-4 days.