DEGs identified in the RNA seq data of 16 h and 7 d treatment groups were further validated by qPCR. Nine candidate genes were selected for q-PCR analysis based on their statistical significance in the RNA-seq data set and on the basis of published literature providing evidence of their involvement in glaucoma pathophysiology12 ,15 ,17 (link),20 (link),23 (link),27 (link),40 (link),51 . The list of primers for the selected genes for validation by qPCR is shown in the Supplementary Table S2 . The expression of significantly altered differentially expressed genes identified in 16 h RNA seq data was validated by qPCR as described previously51 . For 7 d treatment group, qPCR was performed using ABI-QuantStudio 5 (Applied Biosystems, MA, USA).
All mRNA were measured at CT threshold levels and normalized with the average CT values of a reference gene; GAPDH (for 16 h treatment). Values were expressed as fold increase over the corresponding values for control by the 2−ΔΔCT method. The expression of genes in DEX treated HTM cells in logFC ratio was calculated by normalizing with reference control (ACTB; 7 d treatment) and vehicle control.
All mRNA were measured at CT threshold levels and normalized with the average CT values of a reference gene; GAPDH (for 16 h treatment). Values were expressed as fold increase over the corresponding values for control by the 2−ΔΔCT method. The expression of genes in DEX treated HTM cells in logFC ratio was calculated by normalizing with reference control (ACTB; 7 d treatment) and vehicle control.
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