N system allows high-throughput KN-93 (phosphate) biological activity analysis of proteins (and their genes), which can be classified according to families and subfamilies, molecular functions, biological processes, and pathways.Quantitative real-time polymerase chain reactionThe real-time PCR was implemented in an ABI PRISM 7900 sequence detector (Applied Biosystems) in 384-well plates. For the qRT-PCR, total RNA was isolated from the retinas of a second set of animals, because there was insufficient RNA from the microarray experiments for both experiments. Five rats with IOP elevation and five animals with a normal IOP were used for qRT-PCR experiments. One microgram of total RNA was first reverse transcribed using the Omniscript Reverse Transcriptase (5 mM dNTPs, 106RT buffer, 10 units/ml RNase inhibitor, and 10 mM Oligo-dT primer; MWG Biotech) in a total volume of 20 ml for 1 h at 37uC. The enzyme was buy KN-93 (phosphate) inactivated by heating at 95uC for 5 min. The cDNA was diluted twofold, and a 1-ml aliquot was used for each 20-ml PCR using the TaqMan Universal PCR Master Mix and Assays-on-Demand (Applied Biosystems). Assayson-Demand gene expression products consisted of a 206 mix of unlabeled PCR primers and a TaqMan MGB probe (labeled with FAM-TAMRA dye), and they were used to quantify the expression of seven genes: aA crystallin (cryaA), aB crystallin (cryaB), bB1 crystallin (crybb1), crybb2, crybb3, and bA4 crystallin (crybb4), with the 18S RNA (Assay Hs99999901_s1) gene serving as an endogenous control. The assays are designed for the detection and quantitation of specific rat genetic sequences in RNA samples converted to cDNA. The reaction componentsMicroarraysRetinal samples obtained from normotensive (n = 3) and hypertensive (n = 3) rats 4 weeks after IOP elevation were used for the microarray analysis. Animals were killed in a carbon dioxide chamber, and their eyes were immediately enucleated and placed on ice. The retina was removed quickly and collected in RLT buffer, a component of the 1676428 RNeasy kit (Qiagen, Hilden, Germany). A minimum of 10 mg of total RNA/retina was isolated using the RNeasy kit using the procedure described in the manufacturer’s instructions. Total RNA was then shipped on dry ice to MWG Biotech (Ebersberg, Germany), where an aliquot of the RNA was subjected to quality analysis using the 2100 Bioanalyzer system. The RNA was then amplified with T7 polymerase following reverse transcription into cDNA, during which fluorescence-labeled nucleotides (Cy3/Cy5) were incorporated. The labeled probes were hybridized to 24786787 10 k chips (MWG Biotech). Three separate hybridizations per group were carried out with cDNA derived from three separate animals. The 10-k chip consists of 9715 rat genes (5535 Rat 5 k genes) spotted onto one array with an additional 4180 annotated open reading framesProtein Changes in NeurodegenerationFigure 2. Densities of retinal ganglion cells (RGCs) labeled with 5 FluoroGold 8 weeks after the induction of elevated IOP. A RGCs in a normotensive, sham-treated retina. B RGCs in an untreated retina with elevated IOP without hypotensive treatment. C RGCs in a retina with elevated IOP treated with 0.5 timolol (Ti) and travoprost (Ti/Tr) for 4 weeks [the results were the same for those treated with 0.5 Ti and dorzolamide (Ti/D), and 0.5 Ti and brimonidine (Ti/B)]. D Boxplots illustrating the total RGC survival within the different experimental groups. IOP elevation significantly decreased the number of RGCs (*p,0.05) compared with the normotensive, sh.N system allows high-throughput analysis of proteins (and their genes), which can be classified according to families and subfamilies, molecular functions, biological processes, and pathways.Quantitative real-time polymerase chain reactionThe real-time PCR was implemented in an ABI PRISM 7900 sequence detector (Applied Biosystems) in 384-well plates. For the qRT-PCR, total RNA was isolated from the retinas of a second set of animals, because there was insufficient RNA from the microarray experiments for both experiments. Five rats with IOP elevation and five animals with a normal IOP were used for qRT-PCR experiments. One microgram of total RNA was first reverse transcribed using the Omniscript Reverse Transcriptase (5 mM dNTPs, 106RT buffer, 10 units/ml RNase inhibitor, and 10 mM Oligo-dT primer; MWG Biotech) in a total volume of 20 ml for 1 h at 37uC. The enzyme was inactivated by heating at 95uC for 5 min. The cDNA was diluted twofold, and a 1-ml aliquot was used for each 20-ml PCR using the TaqMan Universal PCR Master Mix and Assays-on-Demand (Applied Biosystems). Assayson-Demand gene expression products consisted of a 206 mix of unlabeled PCR primers and a TaqMan MGB probe (labeled with FAM-TAMRA dye), and they were used to quantify the expression of seven genes: aA crystallin (cryaA), aB crystallin (cryaB), bB1 crystallin (crybb1), crybb2, crybb3, and bA4 crystallin (crybb4), with the 18S RNA (Assay Hs99999901_s1) gene serving as an endogenous control. The assays are designed for the detection and quantitation of specific rat genetic sequences in RNA samples converted to cDNA. The reaction componentsMicroarraysRetinal samples obtained from normotensive (n = 3) and hypertensive (n = 3) rats 4 weeks after IOP elevation were used for the microarray analysis. Animals were killed in a carbon dioxide chamber, and their eyes were immediately enucleated and placed on ice. The retina was removed quickly and collected in RLT buffer, a component of the 1676428 RNeasy kit (Qiagen, Hilden, Germany). A minimum of 10 mg of total RNA/retina was isolated using the RNeasy kit using the procedure described in the manufacturer’s instructions. Total RNA was then shipped on dry ice to MWG Biotech (Ebersberg, Germany), where an aliquot of the RNA was subjected to quality analysis using the 2100 Bioanalyzer system. The RNA was then amplified with T7 polymerase following reverse transcription into cDNA, during which fluorescence-labeled nucleotides (Cy3/Cy5) were incorporated. The labeled probes were hybridized to 24786787 10 k chips (MWG Biotech). Three separate hybridizations per group were carried out with cDNA derived from three separate animals. The 10-k chip consists of 9715 rat genes (5535 Rat 5 k genes) spotted onto one array with an additional 4180 annotated open reading framesProtein Changes in NeurodegenerationFigure 2. Densities of retinal ganglion cells (RGCs) labeled with 5 FluoroGold 8 weeks after the induction of elevated IOP. A RGCs in a normotensive, sham-treated retina. B RGCs in an untreated retina with elevated IOP without hypotensive treatment. C RGCs in a retina with elevated IOP treated with 0.5 timolol (Ti) and travoprost (Ti/Tr) for 4 weeks [the results were the same for those treated with 0.5 Ti and dorzolamide (Ti/D), and 0.5 Ti and brimonidine (Ti/B)]. D Boxplots illustrating the total RGC survival within the different experimental groups. IOP elevation significantly decreased the number of RGCs (*p,0.05) compared with the normotensive, sh.