epithelial adherent cell line established from a human colon adenocarcinoma
German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany
The cells were cultured in DMEM containing 20% foetal bovine serum, 100 U/ml penicillin and 100 ng/ml streptomycin. Before passaging, the adherent cells were rinsed with PBS (37°C) three times to remove the fetal bovine serum that inhibits the trypsinization and trypsinized with 5-8 ml of Trypsine/EDTA solution at 37°C in the incubator. Confluent cell cultures (3-6 x 105 cells/cm2) were split, and seeded out for stimulation at 1 x 105 cells/cm2.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. Cells were lysed in an appropriate volume of Buffer RLT. To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA for expression screening.
α33P-dCTP random hexamer labeling
200 - 500 ng mRNA was isolated from total RNA sample using the Oligotex kit (Qiagen) according to the manufacturer’s instructions. Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983). Reversed transcribed cDNA were prepared in a final volume of 30microL as described in detail previously (labeling reaction at 42°C for 60 min; Eickhoff et al. 2000). RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl. Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C. These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction. Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm). The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL). The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C. Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C. Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.
Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan).
Comparison of gene expression patterns of TNF-alpha stimulated and not stimulated CaCo-2 cells
The image data files were gridded using AIDA software (Raytest, Straubenhardt, Germany) and the results were imported in a custom-made database. The ranked expression data distribution was found to follow Zipf´s law (Hoyle et al, 2002) and the logarithmic normal transformed datasets were normalised following this principle, as previously described by Lu et al. 2005. Any hybridisation signal that had a value that fell below a predefined threshold (mean background + 2x standard deviation) were considered as indistinguishable from background and was set to zero. Those values where duplicate expression values for the same gene were not similar (outliers or false positive, difference between duplicates >20%) were eliminated.