Scleral samples were homogenized and then total RNA extracted using a miRVANA™ isolation kit (Ambion Inc.), which is designed to preserve the small RNAs.
Label
Cy3
Label protocol
miRNA profiling experiments were processed according to standard operating procedures in a GLP-compliant setting. Briefly, total RNA was first dephosphorylated and then the pCp-Cy3 labeling molecule ligated to the 3’ end of the RNA molecules.
Hybridization protocol
The labeled RNA was purified using BioSpin6 (Bio-Rad, Hercules CA). Hybridization, washing, staining, imaging, and signal extraction were performed according to Agilent-recommended procedures
Scan protocol
The labeled RNA was purified using BioSpin6 (Bio-Rad, Hercules CA). Hybridization, washing, staining, imaging, and signal extraction were performed according to Agilent-recommended procedures
Data processing
For each probe, the contribution of signal due to background was estimated and removed by the Agilent Feature Extraction software as part of the data file output. Detection calls also made use of the Agilent Feature Extraction software. All other analyses were performed in R (http://www.r-project.org). Arrays within a specific experiment were normalized together according to the Variance Stabilization and Normalization (VSN) method.
