Cultured human trabecular meshwork cells (passage 3) obtained from a donor with no known history of glaucoma. 25y/female/caucasian. Cause of death: pneumonia.
Extracted molecule
total RNA
Extraction protocol
Total RNA from dissected TM tissues and RNA later-fixed cultured HTM cells at passage three was isolated using RNeasy kit (Qiagen Inc., Valencia, CA) following the manufacturer’s protocol. After DNase treatment, RNA yields were determined using the RiboGreen® fluorescent dye (Molecular Probes Inc, Eugene, OR). RNA quality was confirmed using the Agilent 2100 Bioanalyzer.
Label
Biotin
Description
RNA samples were amplified using the OvationTM Biotin RNA amplification and Labeling System (NuGEN Technologies Inc., San Carlos, CA) according to the manufacturer’s instructions. Briefly, total RNA (20 ng) was reverse transcribed into cDNA using a reverse transcriptase and a DNA/RNA chimeric primer. This strand is copied by a DNA polymerase with reverse transcriptase activity to give double-stranded cDNA with an RNA-DNA heteroduplex at one end. This double-stranded cDNA is amplified using the novel isothermal linear amplification method Ribo-SPIA. The cDNA amplification products were fragmented and labeled to generate biotinylated cDNA targets. Biotinylated cDNA targets were hybridized on Affymetrix Human Genome U133 Plus 2.0 high density microarrays following the manufacturer’s instructions.
