RNA was extracted from cells with the use of RNA PLUSTM (Qbiogen, Illkirch, France) and was precipitated with ethanol
Label
biotin
Label protocol
Total RNA (2ug) was amplified and biotin-labelled by a round of in vitro transcription using a MessageAmp aRNA kit (Ambion, Austin, Texas, USA)
Hybridization protocol
Biotin-labelled aRNA (10 ug) was fragmented using 5ug of fragmentation buffer in a final volume of 25 ul, then mixed with 240 ug of Amersham hybridization solution (GE Healthcare Europe GmbH, Freiburg, Germany) and injected on Codelink Uniset I Bioarrays containing around 20,000 human oligonucleotide geneprobes (GE Healthcare Europe GmbH, Freiburg, Germany). The arrays were hybridized overnight at 37°C at 15g in an incubator, then washed in stringent TNT buffer at 46°C for 1 h before performing streptavidin-cy5 (GE Healthcare) detection step.
Scan protocol
The arrays were scanned using a Genepix 4000B scanner (Axon, Union City, CA, USA) and Genepix software, with a laser set at 635 nm, the laser power at 60% and the photomultiplier tube voltage at 60%.
Data processing
The scanned image files were analyzed using CodeLink expression software, version 4.2 (GE Healthcare), which produces both a raw and a normalized hybridization signal for each spot on the array. CodeLink software was used to normalize the raw hybridization signal on each array to the median of the array (median intensity is 1 after normalization) for better cross-array comparison. The threshold of detection was calculated using the normalized signal intensity of the 100 negative control samples in the array: spot with signal intensity below this threshold was referred as ‘absent’. The quality of processing was evaluated by generating scatter plots of positive signal distribution.
gene expression profiling in CD69 positive blood cells selected from Healthy blood Donors and HTLV-1-infected patients
Data table header descriptions
ID_REF
Raw_intensity
The spot intensity, which is the difference between the spot mean and the local background median.
VALUE
The normalized intensity value (the raw intensity divided byt the normalization factor).
Quality_flag
The metrics used to indicate the quality of a spot : G – The spot has passed all quality control measure and is defined as good. C, I, L, M and S indicate presence of issues.
