The adult mouse retina samples were obtained from a pool of C57/B6 mice (n = 350), which were purchased from Charles River Laboratories (Charles River Laboratories, Inc., Willmington, MA). All samples were placed in TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at -80ºC before RNA extraction. Total RNA was extracted with TRIzol reagent according to the manufacturer's instructions. For a quality control measure of the samples, total RNA was ran through a 1% agarose gel and using the 2100 Agilent BioAnalyzer System (Agilent Technologies, Palo Alto, CA) to check for possible contamination and degradation.
Biomaterial provider
Charles River Laboratories
Extracted molecule
total RNA
Label
Biotin-11-UTP
Label protocol
The ExpressChip MO3 DNA Microarray System (Mergen Ltd., San Leandro, CA) was used for this study. The assay was carried out according to the manufacturer's instructions. In brief, DNase-treated total RNA (20 µg) was reverse-transcribed using an oligo[(dT)24 T7 promoter]65 primer (consisting of the nucleotide binding sequence for the T7 RNA polymerase followed by 24 thymidine nucleotides) followed by second strand synthesis. ). The double-stranded cDNA was cleaned using the Qiaquick purification kit (Qiagen) all of which was used as a template for In Vitro Transcription (IVT) utilizing Ambion's T7 MEGA script reagents and Biotin-11-UTP (PerkinElmer/NEN).
Hybridization protocol
These probes were hybridized to the arrays overnight at 30°C with continuous agitation. The arrays were then washed, and hybridized probes were detected using a streptavidin/cyanine-3 fluorescent dye-conjugated antibody.
Scan protocol
Chips were imaged using a ScanArray scanner (PerkinElmer, Boston, MA).
Description
MRP1 technical replicate 2 (site 2)
Data processing
Signal intensities across microarrays were normalized using the quantile normalization (www.bioconductor.org).
