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.
The mouse cortex samples were obtained from P1 C57/B6 mice (n = 19), which were purchased from Charles River Laboratories (Charles River Laboratories, Inc., Willmington, MA).The mouse cortex was used as a reference sample. Universal mRNA reference was still being developed when the study started, so it was not utilized. 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.
Extracted molecule
total RNA
Label
Cy3
Hybridization protocol
The Agilent Fluorescent Linear Amplification Kit (Agilent Technologies) was used for the preparation of fluorescently labeled target samples. Briefly, both first and second strand cDNAs were synthesized by incubating 3 µg of total RNA with T7 promoter primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3') using SuperScript II (Invitrogen, Carlsbad, CA). Fluorescently labeled complementary RNA (cRNA) was amplified by adding 4 ?l of cyanine 3-dCTP (6 mM) or 4 µl of cyanine 5-dCTP (4 mM) (PerkinElmer, Inc., Boston, MA) to the 20 ul of cDNA template and mixing with 56 µl of reaction mixture containing, in the following order: 20.1 µl of nuclease-free water, 20 µl of 4X transcription buffer, 6 µl of 0.1 M DTT, 8 µl of NTP mix, 0.5 µl of RNaseOUT, 0.6 µl of inorganic pyrophosphatase, and 0.8 µl of T7 RNA polymerase. After incubating at 40°C for 3 hr, cRNAs were cleaned up using RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Five ug of each labeled cRNA target were used for hybridization on the oligonucleotide-arrayed slide after being fragmented by fragmentation buffer (Agilent Technologies). After overnight hybridization at 65°C, the oligonucleotide arrays were washed and scanned by the Agilent Scanner G2505A (Agilent Technologies).
