Primary astrocytes were obtained from 1-day old Sprague Dawley rats as previously described in Shin et al. (2001), Immunostimulation of rat primary astrocytes decreases intracellular ATP level. Brain Res., 902, 198-204.
Extracted molecule
total RNA
Extraction protocol
QIAGEN RNeasy, according to manufacturer
Label
biotin
Label protocol
RNA samples were labeled according to the chip manufacturers recommended protocols. In brief, 500ng of high-quality total RNA was amplified and labeled with TotalPrep amplification kit (Ambion, Austin, TX). Total RNA was reverse transcribed with oligo(dT) primer, and second strand was synthesized. Purified cDNA was in-vitro transcribed with T7 polymerase to generate antisense RNA copies of each mRNA in a sample. Biotin-UTP are incorporated into cRNA during the in-vitro transcription.
Hybridization protocol
750ng of amplified cRNA was hybridized to RatRef-12 expression beadchip (Illumina, San Diego, CA) for 16 hours.
Scan protocol
The hybridized array was detected with streptavidin-Cy3 (Amersham Biosciences, UK) and scanned with the BeadArray Reader (Illumina).
Description
Scanned raw data ([.tiff] image files and [.csv] probe location info. files) were processed with a R package, 'Beadarray'.
Data processing
The column VALUE in the table was obtained by quantile normalization of log2(SIGNAL-BACKGROUND) and used for all the analysis (Bolstad, B. M., Irizarry R. A., Astrand, M., and Speed, T. P. (2003), A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19, 185-193).
