Umbilical cord tissue (300 mg) was first placed in a sterile Dispomix tube and homogenized for 55s for 3 cycles in 3 ml of Trizol using the Dispomix (Medic Tools, AG, Zug, Switzerland). After spinning down the debris, the supernatants were divided equally into three 2ml tubes. 200ul of chloroform were added to each tube, vortexed vigorously and centrifuged for 15min at 4˚C. The aqueous phase was carefully transferred to a new tube containing 1ul of linear acrylamide. An equal amount of isopropanol was added and mixed by inversion. After incubating at -20˚C overnight to precipitate the RNA, the pellet was obtained by centrifuging at 13,200 rpm for 10 min at 4˚C. The RNA pellet was washed twice in 70% (v/v) ethanol, air-dried and resuspended in RNase-free water. The isolated RNA was then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). On-column DNase digestion was carried out before the first wash step according to the manufacturer ’s instructions. The purified RNA was then eluted in 30 µl of RNase-free water and stored at -80 °C. RNA concentration and purity were measured using a nanodrop ND-8000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and RNA integrity was determined using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Labchips (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Briefly, Cy3-labelled cRNA was generated from 100 ng of total RNA using the Quick Amp Labelling Kit (One-Color) (Agilent Technologies, Santa Clara, CA, USA).
Hybridization protocol
Hybridization performance was assessed by means of 10 proprietary spike-in controls incorporated into the cRNA synthesis procedure. The labelled cRNA was then purified and hybridized onto Agilent SurePrint G3 Human Gene Expression (8 x 60 K) microarrays in a rotating (10 rpm) hybridization oven for 17 h at 65°C, after which they were washed and processed with proprietary buffers and solutions.
Scan protocol
The microarrays were then scanned at a resolution of 3 µm on an Agilent scanner using an extended dynamic range (PMT 10/100). The image data were processed using default values in feature extraction version 10.7.1.1 (Agilent Technologies, Santa Clara, CA, USA).
Description
Group: HBW
Data processing
Agilent “.txt” files were outputted from the scanner and loaded into Arraystudio (Omicsoft). Signal extraction was performed from the gProcessedSignal value incorporating background subtraction. All expression values were log transformed. All probes with expression levels less than two standard deviations above background were removed. Values across replicate probes were averaged. Data were normalised amongst samples using quantile normalisation. Two samples with MAD scores <-5 were removed from the analysis. Duplicate data for the same sample were averaged.
