For normalization, CT values were calculated as CT (gene of interest) C geometric mean of CTs for the normalizers

For normalization, CT values were calculated as CT (gene of interest) C geometric mean of CTs for the normalizers. = 11). Two control groups were also included: 10 eyes from 10.5-month-old DBA/2J-control group (dataset 1, Figure ?Physique1A).1A). For the ONH, 223 and 456 probe units were DE ( 0.05) in the NOE1 and NOE2 groups, respectively, compared with control. Importantly, the vast majority of probes DE by at least 2-fold were the same in these impartial groups. The two most DE genes were lipocalin 2 (control samples was performed using the gene expression values of 570 disease-relevant probe units (Methods and Supplemental Physique 1; supplemental material available online with this short article; doi: 10.1172/JCI44646DS1). Hierarchical clustering first linked the most comparable eyes, and continued until Ancarolol all eyes were linked in a dendrogram (Physique ?(Physique1B1B and Methods). As the least comparable eyes were linked at the highest branch points, a cutoff (threshold of relatedness) was applied to separate groups of eyes into impartial disease stages (Methods). We selected a cutoff that made biological sense based on standard morphological criteria, e.g., controls were grouped into their own stage, and SEV eyes were individual from NOE eyes. We only considered groups PVR made up of at least 4 eyes as a stage. Using these criteria, 5 new, molecularly defined, stages of glaucoma were recognized (dataset 2, stage 1 to stage 5; Physique ?Physique1,1, B and C). Not surprisingly, 10 of the 40 eyes did not fit into these 5 stages. This displays the large number of possible molecular says for such a variable disease. The 5 stages were ordered based on two criteria: (a) Quantity of DE genes compared with no-glaucoma control: stage 1 experienced the smallest quantity of DE genes when stages 1C5 were compared with the control group (381 DE genes, Physique ?Physique1C)1C) and was considered to be the earliest molecular stage. (b) Previously decided morphological damage: although stages 2C4 had a similar quantity of DE genes (approximately 10,000), only stages 1 and 2 contained eyes with no detectable glaucoma (NOE). In comparison, stage 3 contained 3 eyes with NOE glaucoma and 3 eyes with MOD glaucoma. Ancarolol Stage 4 contained 4 eyes with MOD glaucoma. Therefore, stage 3 was decided to be between stage 2 and stage 4. Stage 5 contained only eyes with SEV glaucoma. As expected, axon number decreased with increasing glaucoma stage (Physique ?(Figure2A).2A). Also, the Ancarolol number of genes whose expression value was DE by at least 2 SDs from the average of controls increased from stages 1 to 5 (Spearmans correlation 0.9, 10C100) (Determine ?(Figure2B). 2B). Open in a separate windows Physique 2 Differences Ancarolol between the molecularly defined ONH stages.(A) Overall, axon number decreased, from stage 1 to 5. However, for stages 1C3, there was no significant axon loss with respect to D2-control eyes ( 0.1). (B) The number of probe sets altered by at least 2 SDs (with respect to D2-control) increased with disease stage. (C) PCA showed that stages 1C5 occupy essentially non-overlapping territories. D2-control samples that did not cluster (open diamonds, upper left quadrant) were clearly unique from DBA/2J glaucoma samples (filled colored diamonds). DBA/2J samples that did not cluster are shown as filled black diamonds. Based on optic nerve damage, 3 of the 6 eyes in stage 3 were indistinguishable from no-glaucoma control eyes. The other 3 eyes experienced MOD glaucoma (Supplemental Physique 2). This indicates that this molecular changes determining.