Ovation pico wta system v2

Fresh monkey anterior eye segments were embedded in OCT and immediately frozen on dry ice prior to storage at −80°C. Twelve-micrometer-thick frozen sections were transferred to glass polyethylene naphthalate foil slides (#11505189; Leica Microsystems, Wetzlar, Germany) as we previously described^{30 (link)} and stained with Eosin Y. TM was dissected using a laser micro-dissection system (LMD 6000 and CTR 6500, Leica Microsystems) (Fig. 3). Some areas of uveal meshwork were included to obtain adequate RNA quantities. RNA extraction was performed using the RNeasy Micro Kit (Qiagen, Hilden, Germany) and amplification with Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA, USA). The quantitative PCR (qPCR) analysis was performed using LightCycler 480 Software according to manufacturer protocols. Quantified values for each gene of interest were normalized against the input determined by the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Actb, NM_007393). We custom designed the primers for the monkey species to detect fibronectin, type IV collagen, and NF-κB (Supplementary Table S1). Each assay was performed in triplicate utilizing mean values for statistical analysis.

For CCL21 experiments, CTR and INF ears from two mice per condition were pooled, enzymatically digested, and FACS-sorted as described above. Extracted and purified RNA (PicoPure RNA isolation kit; Thermo Fisher Scientific) was converted and amplified into cDNA using the Ovation Pico WTA System V2 (NuGen). For talin1 experiments, RNA from LPS mature DCs previously isolated from the bone marrow of Mx1-Cre^+/− Tln1^fl/fl mice was extracted and converted into cDNA. Quantitative PCR analyses of Ccl21 (Ccl21 Mm0364971_m1; ACTB, both Thermo Fisher Scientific) and Tln1 (Tln1 forward: 5′-GGC​CCT​CCC​AAC​GAC​TTT-3′, reverse: 5′-AGC​CTC​TAG​CCA​GAT​GCC​TTT-3′; Rpl0 forward: 5′-AGA​TTC​GGG​ATA​TGC​TGT​TGG-3′, reverse: 5′-TCG​GGT​CCT​AGA​CCA​GTG​TTC-3′) gene expression were performed on a Fast Real-time PCR system (Thermo Fisher Scientific).

Purified total RNAs from 95 tubule samples were amplified using the Ovation Pico WTA System V2 (NuGEN) and labeled with the Encore Biotin Module (NuGEN) according to the manufacturer's protocol. The purified total RNA from 41 tubule samples used for validation were amplified using the Two-Cycle Target Labeling Kit (Affymetrix) as per the manufacturer's protocol. Transcript levels were analyzed using Affymetrix U133A arrays.