tissue: Eye genotype: mmachc_mutant time point: 7 dpf
Extracted molecule
total RNA
Extraction protocol
Trizol (Invitrogen) and RNaseazy (Qiagen) kits were used to extract total RNA according to the manufacturer's instructions.
Label
Biotin
Label protocol
Labeling was performed on a sample-by-sample basis according to manufacturer’s guidelines for use with the Affymetrix Mouse HuGene-1_0-st-v1 GeneChips (Affymetrix, Inc). DNase treatment was included as part of isolation to remove possible contaminating DNA. Bioanalyzer nanochip (Agilent, Inc) and NanoDrop (ThermoFisher, Inc) were used to validate and quantitate the RNA prior to cRNA synthesis and labeling. 300 ng of total RNA was used for labeling with theAmbion WT Expression Kit (cat#4411974) and Affymetrix kit (Cat # 900652).After hybridization, the probe arrays were stained with streptavidin-phycoerythrin solution (Molecular Probes) and enhanced using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories).
Hybridization protocol
The hybridization cocktail containing the fragmented and labeled cDNAs were hybridized to Affymetrix mouse GeneChip 1.0 ST microarrays. The microarrays were washed and stained in an Affymetrix Fluidics Station using standard Affymetrix protocols.
Scan protocol
Genechips were scanned using an Affymetrix Gene Chip Scanner 3000.
Data processing
Gene expression intensities were calculated using GeneChip Command Console Software (AGCC). .cel files generated by the Affymetrix AGCC program were imported in the Affymetrix Expression Console software (4-26-7-53) and RMA (Robust Multichip Analysis) normalization was performed to generate the .chp files. The .chp files were normalized, log2 transformed, and summarized.
