Gfx96 real time system

From human cornea (n = 11), central CE was collected after DM peeling (central 8 mm diameter). The posterior limbus was collected by an inward peeling method (https://www.youtube.com/watch?v=wQlPhHCJgmM&t=121s). Under stereo-microscope, the tissue was further dissected to separate the innermost transparent PE with <0.5 mm width, the outermost pigmented TM tissue, and the remaining intermediate non-pigmented smooth TZ. All samples were washed at least three times in ice-cold PBS, followed by preservation in Trizol reagent (Sigma-Aldrich). Total RNA was extracted using RNeasy kit (Qiagen, Hilden, Germany) and on-column RNase-free DNase kit (Qiagen). After reverse transcription using Superscript III RT-PCR kit (ThermoFisher), cDNA was assayed for candidate gene expression with specific primer pairs (Supplementary Table S3) by quantitative real-time PCR (qPCR) using Sybr Green Supermix (BioRad, Herculus, CA, USA) or Taqman assay in GFX96 real-time system (BioRad). Experiments were run in quadruplicate. The relative gene expression (ΔCT) normalized with mean CT of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CT_GAPDH) or β-actin (ACTB) (CT_ACTB) was obtained, and fold change to the central CE was expressed as mean ± SD.

Samples in RLT buffer (Qiagen, Valencia, CA, USA) or Trizol (Sigma) were processed for total RNA extraction using RNeasy kit (Qiagen) and on‐column RNase‐free DNase kit (Qiagen) according to manufacturer's protocol. Total RNA (1 μg) was reverse transcribed using Superscript III RT‐PCR kit (Invitrogen) with random hexanucleotide primer (10 ng/mL, Invitrogen). Gene expression was assayed with specific primer pairs (Table S3) by quantitative real‐time PCR (qPCR) using Sybr Green Supermix (Bio‐Rad) in GFX96 Real‐time System (Bio‐Rad). Experiments were run in quadruplicate. Relative gene expression of each sample (ΔCT) was normalized by the mean CT of the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (CT_GAPDH) and expressed as mean and standard deviation (SD).

Cells were collected in RLT buffer and RNA was extracted using RNeasy kit (Qiagen, Valencia, CA, US) and on-column RNase-free DNase kit (Qiagen) according to manufacturer’s protocol. In brief, total RNA (1 μg) was reverse transcribed using Superscript III RT-PCR kit (Invitrogen) with random hexanucleotide primer (10 ng/ml, Invitrogen). Gene expression was assayed with specific primer pairs (Supplementary Table 5) by quantitative real-time PCR (qPCR) using Sybr Green Supermix (BioRad) in GFX96 Real-time System (BioRad). Experiments were run in quadruplicate. Relative gene expression of each sample (ΔCT) was normalized by the mean CT of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CT_GAPDH) and expressed as mean and standard deviation (SD). Fold changes comparing expression in SF versus CSK were calculated.