Abi quantstudio 5

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 pathophysiology^{12 ,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.

Custom 96-well miRNA PCR arrays were designed containing 19 miRNAs of interest, 3 endogenous controls (miR-103a-3p, miR-423-5p and miR-191-5p), and 2 positive controls for cDNA synthesis and PCR (UniSp3 and UniSp6, Table S4). RNA was isolated from H1975P and H1975GR (n = 3) using the miRCURY™ RNA isolation kit (Exiqon A/S, Vedbaek, Denmark). First-strand cDNA synthesis and real-time PCR amplification was carried out as per the miRCURY LNA™ Universal RT microRNA PCR protocol (Exiqon A/S). Each assay was run in duplicate wells on an ABI QuantStudio 5 (Applied Biosystems, Foster City, CA, USA). Results were pre-processed and analyzed using the downloadable Exiqon qPCR data analysis software, GenEx.

Real-time fluorescence quantitative PCR was performed using the SYBR^® Premix Ex Taq^TM II kit (Takara). The transcript levels of each gene were measured using ABI QuantStudio 5 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and gene-specific primers (Table 1). The thermal cycling conditions for the PCR were 95 °C pre-denaturation for 30 s, 95 °C for 3 s, 60 °C for 30 s (40 cycles). To ensure primer specificity, the PCR amplification curve and melting curve were confirmed after each operation, and each sample was analyzed three times. The relative transcript level of each gene was calculated using the 2^−∆∆Ct method [28 (link)]. The tilapia β-actin gene was used as an internal reference.