Fam mgb

Retinal tissues were collected using a dissecting microscope at different time points after ONC and stabilized in RNAlater (Qiagen, Valencia, CA). Total RNA was extracted utilizing RNeasy Mini Kit (Qiagen, Valencia, CA) per manufacturer’s instructions. For first-strand cDNA synthesis, a 20ul reverse transcription reaction system containing Superscript III, oligo (dT) and dNTPs mixture was employed. Briefly, the reaction was incubated at 25 °C for 10 minutes, 50°C for 30 minutes, followed by inactivat ion at 85 °C for 5 minutes, then chilled on ice and 1ul of RNase H was added. The reaction was then incubated at 37 °C for 20 minutes. Taqman real-time PCR assay was carried out using 7500 Fast System (ABI 7500; Applied Biosystems, Foster City, CA) in a standard 7500 mode. The thermal cycling condition was as follows: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds, 60 °C for 1 minutes, followed by 55 °C for 10 seconds, fi nally 25 °C for 5 minutes. Mean Ct value was obtained and relative mRNA level was measured by the 2^−ΔΔCt method and normalized using the housekeeping gene GAPDH. The following predesigned gene-specific TaqMan® probe and primer sets were employed: Pdcd1, FAM-MGB (Applied Biosystems, Foster City, CA), GAPDH, FAM-MGB (Applied Biosystems, Foster City, CA).

RNA was isolated from tissue samples with the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. RNA was extracted using the NucleoSpin RNA XS Kit for RNA-seq (Takara Bio) or the ARCTURUS PicoPure RNA Isolation kit (Thermo Fisher Scientific). The High-Capacity RNA-to-cDNA kit (Applied Biosystems) was used for reverse transcription. TaqMan gene expression assays were used to quantify target genes using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and primers for Cxcl12 (Mm00445553_m1), Vcam1 (Mm01320970_m1), Kitl (Mm00442972_m1), Angpt1 (Mm00456503_m1), Il6 (Mm00446190_m1), Vegfa (Mm00437306_m1) and Vcan (Mm01283063_m1, all FAM–MGB, Thermo Fisher Scientific) as well as Gapdh (Mm99999915_g1, VIC–MGB, Thermo Fisher Scientific). Samples were run on a 7500 Real-Time PCR system (Applied Biosystems), and target gene expression was normalized to Gapdh expression.

RNA samples extracted for gene expression profiling were used to proceed with RT-qPCR analysis. cDNA was made using Omisncript RT Kit (Qiagen). Taqman Fast Advanced Master Mix and FAM-MGB primers were purchased from ThermoFisher Scientific. Reactions were conducted on a StepOnePlus Real-Time PCR System (Applied Biosystem).