gender: male age: 8.9 weeks tissue: retina treatment: CNTF injected genotype: control
Treatment protocol
Five CNGB3-mutant dogs (carrying the D262N missense mutation; CNGB3m/m) and 5 normal control (homozygous: CNGB3+/+; heterozygous CNGB3+/m) dogs were injected unilaterally with 12 µg of intravitreal CNTF (OD eyes) and the contralateral eyes (OS) served as controls and were injected with PBS. Retinas were collected at 1 week post injection.
Growth protocol
Samples were obtained from dogs of a research colony with a common genetic background that were maintained at the Retinal Disease Studies Facility (RDSF) in Kennett Square, Pennsylvania. Both eyes were enucleated following intravenous anesthesia with sodium pentobarbital, and the dogs were euthanatized with an overdose of euthanasia solution (Euthasol; Virbac, Fort Worth, TX). Retina and choroid samples were immediately collected, flash frozen in liquid nitrogen, and stored in -80°C until use. The research was conducted in full compliance with the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from dog retinas following standard procedures of the manufacturer (miRNeasy minikit; Qiagen Inc, Valencia, CA). RNA concentration was quantified with a NanoDrop-1000 Spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Wilmington, DE) and RNA quality was verified by microcapillary electrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA with RIN> 8 and A260/280 ratio> 1.9 was used in both microarray and the following qRT-PCR analyses.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng total RNA using the Low Input Quick Amp WT Labeling Kit, One-Color, according to the manufacturer's instructions (Agilent), followed by RNAeasy mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yields were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
A total of 1.65 µg Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. After completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the canine Agilent Oligo Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed one minute at room temperature with GE Wash Buffer 1 (Agilent) and one minute with GE Wash Buffer 2 (Agilent) pre-heated at 37°C, and finally dried by brief centrifugation.
Scan protocol
Following the washing step, microarray slides were scanned immediately with a GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA).
Description
genotype: CNGB3+/m
Data processing
The scanned images were analyzed with Feature Extraction Software (version 9.5.3.1, Agilent) using default parameters to obtain background subtracted and spatially detrended raw data (gProcessedSignal values). Feature non-uniform outliers flagged in Feature Extraction were excluded from further analyses. The Partek Genomics Suite 6.4 software (Partek Incorporated, St. Louis, MO) was used for all statistical analyses; specifically it was used to extract the gProcessedSignal values from Agilent, log2 transform and quantile normalize them. A one-way ANOVA was then applied to compare gene expression of CNGB3-affected vs. normal retinas in either the PBS or the CNTF injected eye, while a 2-way ANOVA was used to compare CNTF vs. PBS injected eyes in either normals or CNGB3-affected dogs. Genes with Benjamini Hochberg (BH)-adjusted (stepup) p < 0.05 and fold change (FC) > +/-2 were considered statistically significant differentially expressed (DE).
