Cells were grown to confluence prior to the experiments. Millimolar solutions of ascorbic acid (AA) were prepared using PBS (pH 7.4) immediately prior to the experiments. Solutions were sterilised by passing through a 0.22 µm membrane and protected from direct light. The solutions were then diluted in pre-warmed medium to obtain the desired concentrations and added to the cells. Post-confluent HDFs were supplemented with 100 µM AA added fresh every day for a period of 5 days.
Growth protocol
Cells were grown as a monolayer culture in Nunclon culture flasks (Invitrogen, Paisley, UK) at 37 °C in a humidified atmosphere containing 5 % CO2. Cells were grown in MEM with Earle’s salts containing non-essential amino acids supplemented with 2 mM Glutamax-I and 10 % (v/v) FBS. Cells were passaged when nearly confluent. For passaging, growth medium was removed and cells were washed in pre-warmed sterile phosphate buffered saline (PBS). Cells were incubated in PBS containing 0.05 % (w/v) porcine trypsin and 0.02 % (w/v) tetrasodium EDTA at 37 °C for 2 minutes or until cells started to separate. The flask was then gently tapped to help release the cells. Once cells were floating, growth medium was added to the flasks to inactivate the trypsin and cells were centrifuged at 300 × g for 4 minutes. With the supernatant removed, the tube was gently tapped to disaggregate the pellet and the cells were resuspended in an appropriate volume of growth medium and distributed into new flasks (split ratio 1:3).
Extracted molecule
total RNA
Extraction protocol
T-RNA was extracted using TRI Reagent (Sigma, Poole, UK), according to the manufacturer’s instructions. All additional reagents used were of molecular biology grade and purchased from Sigma (Poole, UK). Briefly, cells were lysed directly in the culture dish with 1 ml TRI Reagent and the resulting cell lysate passed several times through a pipette to achieve homogenisation. The homogenised samples were incubated in microcentrifuge tubes at room temperature for 5 minutes to permit the complete dissociation of nucleoprotein complexes. 200 µl of chloroform were added to each homogenised sample, tubes shaken vigorously by hand for 15 seconds and incubated at room temperature for 2 to 3 minutes. Samples were centrifuged at 12,000 × g for 15 minutes at 4 °C, after which the aqueous phase was transferred to a fresh tube. RNA was precipitated from the aqueous phase by mixing with 0.5 ml isopropyl alcohol. Samples were incubated at room temperature for 10 minutes and centrifuged at 12,000 × g for 10 minutes at 4 °C. Supernatant was removed and RNA pellet washed once with 1 ml of 75 % ethanol. Samples were mixed by vortexing and centrifuged at 7,500 × g for 5 minutes at 4 ºC. At the end of the procedure, the RNA pellet was briefly air-dried for 5 to 10 minutes, after which RNA was dissolved in an appropriate volume of RNase/DNase-free water, pre-warmed to 60 ºC, by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60 ºC. A second RNA cleanup was performed using the QIAGEN RNeasy Mini kit according to the manufacturer’s instructions. T-RNA concentration was determined by measuring the absorbance of a 1:100 diluted solution at 260 nm in a GeneQuant II RNA/DNA calculator spectrophotometer (Pharmacia Biotech, Cambridge, UK), assuming that if A260 = 1, the content equals 40 µg/ml of RNA. T-RNA integrity was investigated in the Agilent 2100 Bioanalyzer, by assessing size distribution of the T-RNA using the Eukaryote Total RNA Nano assay (Agilent Technologies, Palo Alto, California, USA), according to the manufacturer’s specifications.
Label
biotin
Label protocol
The One-cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, California, USA) was used to convert the mRNA in the T-RNA samples into ds-cDNA by reverse transcription with an oligo-dT primer. Initially, first strand cDNA was formed in a reaction containing 6 g of T-RNA, 2 µl of diluted poly-A RNA controls, 2 µl of 50 µM T7-oligo (dT) primer and RNase-free water to a final volume of 12 µl. This mixture was incubated at 70 °C for 10 minutes and chilled at 4 °C for 5 minutes, after which the following reagents were added: 4 µl of first strand reaction mix, 2 µl of 100 mM dithiothreitol (DTT) and 1 µl of 10 mM dNTP. Tubes were placed at 42 °C for 2 minutes to equilibrate the temperature prior to the addition of Superscript II reverse transcriptase (1µl) and further incubated for 1 hour at 42 °C. The second strand cDNA synthesis was performed by adding, to the solution containing the first strand cDNA, 3 µl of 10mM dNTP, 1 µl of E. coli DNA ligase, 4 µl of E. coli DNA polymerase I, 1 µl of E. coli RNase H, 30 µl of second strand reaction buffer and 91 µl of RNase-free water. The reaction was incubated for 2 hours at 16 °C. Following this incubation, 2 µl of T4 DNA polymerase were added and incubated for 5 minutes at 16 °C. The reaction was stopped by the addition of 10 µl of 0.5 M EDTA. The resulting ds-cDNA was cleaned up using the Affymetrix Sample Cleanup Module, according to the manufacturer’s instructions. Synthesis of biotin labelled cRNA was performed by in vitro transcription using the Affymetrix IVT labelling kit. Briefly, the ds-cDNA synthesised above (12 µl) was used as template, and added to 4 µl of 10× IVT labelling buffer, 12 µl of Labeling NTP mix, 4 µl of IVT Labeling Enzyme mix and 8 µl of RNase-free water. The reaction was incubated at 37 °C for 16 hours. This step was followed by cRNA cleanup with the Affymetrix Sample Cleanup Module, according to the manufacturer’s specifications. Quantification of cRNA was performed in a spectrophotometer by measuring the absorbance at 260 nm and the adjusted cRNA yield calculated taking into account the carryover of unlabelled T-RNA by the following formula: adjusted cRNA yield = RNAm – (T-RNAi)(y), where RNAm is the amount of cRNA measured after the IVT reaction, T-RNAi is the starting amount of T-RNA and y is the fraction of cDNA used in the IVT reaction. Size distribution of the labelled transcripts was investigated in the Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Nano assay (Agilent Technologies, Palo Alto, California, USA). The appropriate amount of cRNA (20 µg) was fragmented by adding 8 µl of the fragmentation buffer provided in the Sample Cleanup Module and RNase-free water to a final volume of 40 µl, and incubating at 94 °C for 35 minutes. Fragmentation efficiency was confirmed by analysing the size distribution of the fragmented, labelled transcripts in the Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Nano assay.
Hybridization protocol
Fragmented, labelled cRNA (15 µg) was added to 15 µl of 2× hybridisation buffer (final 1× concentration is 100 mM 2-Morpholinoethanesulfonic acid (MES), 1 M [Na+], 20 mM EDTA, 0.01% Tween 20), 3 µl of 10 mg/ml herring sperm DNA (Promega, Southampton, UK), 3 µl of 50 mg/ml acetylated bovine serum albumin (BSA) (Invitrogen, Paisley, UK) and 30 µl of DMSO. The hybridisation buffer was spiked with 5 µl of 3nM control oligonucleotide B2 and 15 µl of the 20× eukaryotic cRNA controls bioB, bioC, bioD and cre (final concentration is 1.5, 5, 25 and 100 pM, respectively), all controls were from the GeneChip Eukaryotic Hybridisation Control Kit (Affymetrix). The hybridisation mixture was heated to 99 °C for 5 minutes, cooled to 45 °C for 5 minutes and centrifuged at 14,000 × g for 5 minutes to remove any insoluble material from the hybridisation mixture. Each hybridisation mixture containing 10 µg of fragmented biotin-labelled cRNA (200 µl) was hybridised to a pre-wetted (with hybridisation buffer) GeneChip Human U133 Plus 2.0 array (Affymetrix) for 16 hours at 45 °C with permanent rotation at 60 rpm in a Affymetrix Hybridisation Oven 640. Probe array washing and staining were performed according to EukGE-WS2v5 Gene Chip protocol (Affymetrix) in the Affymetrix Fluidics Station 400. Briefly, arrays were washed in a non-stringent wash buffer containing 6× SSPE (0.9 M NaCl, 0.06 M NaH2PO4, 0.006 M EDTA) (Cambrex, Berkshire, UK) and 0.01% Tween-20 at 25 °C, then with a stringent buffer containing 100 mM MES, 0.1 M [Na+], 0.01% Tween-20 at 50 °C. Following this wash, the probe arrays were stained with a solution containing 10 µg/ml of streptavidin-phycoerythrin (SAPE) conjugate (Invitrogen, Paisley, UK) and 2 mg/ml acetylated BSA (Invitrogen) dissolved in a buffer containing 100 mM MES, 1 M [Na+], 0.05% Tween 20. Following staining, the probe arrays were exposed to 3 µg/ml goat biotinylated anti-streptavidin antibody (Vector laboratories, Peterborough, UK) in a buffer as above containing 0.1 mg/ml normal goat IgG (Sigma, Poole, UK) and 2 mg/ml acetylated BSA (Invitrogen). Finally, the probe arrays were re-stained with SAPE as described above.
Scan protocol
Probe arrays were scanned once at 570 nm using an Affymetrix GeneChip scanner and the fluorescence intensity of the scanned image registered in CEL intensity files.
Description
n/a
Data processing
DNA-Chip Analyser (dChip) software (Li and Wong 2001 PNAS, 98:31) was used to automatically select probes, detect outliers/artefacts, normalise the fluorescence intensity over multiple arrays and calculate model-based expression values from CEL intensity files using the PM/MM values.
