Visual form deprivation (VFD) was induced in 12 mice by applying a unilateral frosted hemispherical plastic diffusers over the right eyes. Briefly, frosted hemispherical plastic diffusers were hand-made using caps from 0.2 ml PCR tubes (Molecular BioProducts, San Diego, CA) and rings made from medical tape (inner diameter 6 mm; outer diameter 8 mm). A cap was frosted with fine sandpaper and attached to a ring with Loctite™ Super Glue (Henkel Consumer Adhesives, Avon, OH). On the first day of the experiment (P24), animals were anesthetized via intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg), and diffusers were attached to the skin surrounding the right eye with several stitches using size 5-0 ETHILON™ microsurgical sutures (Ethicon, Somerville, NJ) and reinforced with Vetbond™ glue (3M Animal Care Products, St. Paul, MN). The contralateral untreated left eyes were used as control. Toenails were covered with adhesive tape to prevent mice from removing the diffusers. Animals recovered on a warming pad and were then housed in transparent plastic cages for the duration of the experiment (10 days). A control group comprised of 8 age-matched C57BL/6J mice was maintained under the same experimental conditions as experimental group, but without VFD.
Extracted molecule
total RNA
Extraction protocol
Both myopic and control eyes were enucleated, cleaned by removing surrounding tissues and the crystalline lens, retina and sclera were dissected, snap-frozen in liquid nitrogen and stored in RNAlater®-ICE (Life Technologies, Grand Island, NY). In order to obtain sufficient amount of tissue for RNA isolation, the retinas or scleras from three myopic or control eyes were pooled for RNA extraction. Three replicates (3 eyes per replicate) were processed in parallel. Total RNA was isolated from the retina and sclera samples using mirVanaTM miRNA isolation kit (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. RNA concentration was measured using NanoDrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE). The quality of the total RNA was assessed using Agilent RNA 6000 Nano kit and 2100 Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) following the manufacturer’s instructions.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng of total RNA using the miRNA Complete Labeling and Hyb Kit (Agilent) according to the manufacturer's instructions.
Hybridization protocol
Microarray hybridization was carried out using miRNA Complete Labeling and Hybridization Kit (Agilent) followed by washing using Gene Expression Wash Buffer Kit following manufacturer's instructions.
Scan protocol
Microarrays were scanned on a DNA microarray scanner (Agilent G2565BA) and features were extracted using the Agilent Feature Extraction (AFE) image analysis tool (version A.9.5.3) with default protocols and settings.
Description
C1_Sclera miRNA expression, control sclera.
Data processing
Gene expression data were analyzed using Partek Genomics Suite 6.6. Data were adjusted to bring the minimal signal to 0.5, normalized using quantile normalization procedure, and log2-transformed. This was followed by the removal of absent features and outliers. The normalized data were then analyzed using ANOVA to identify the differences in miRNA expression levels between myopic and control eyes. Differentially expressed miRNAs were identified using an FDR-adjusted P-value threshold of 0.05 and a cutoff of 2-fold change in expression. Differential expression was calculated as fold change (FC, myopic samples vs control).
