Random primer hexanucleotides

Cells were collected in RLT buffer (Qiagen) with freshly added β-mercaptoethanol (β-ME; Sigma). Total RNA was extracted using RNeasy Miniprep kit (Qiagen) with on-column RNase-free DNase kit (Qiagen), following manufacturers’ instructions. After quantification by Nanodrop (Thermo Fisher), total RNA (500 ng) was reverse-transcribed with SuperScript III Reverse Transcriptase kit (Thermo Fisher) and random primer hexanucleotides (10 ng/ml, Thermo Fisher). Complementary DNA (cDNA) was assayed for gene expression using SYBR Green Real-Time Master Mix (Life Technol, Carlsbad, CA) and specific primer (Additional file 1: Table S2) in a QuantStudio 3 real-time PCR system (Applied Biosystems, Thermo Fisher). The experiments were run in triplicate. The relative RNA abundance was determined by ΔCT method after normalization with the housekeeping 18S. A similar protocol was followed for mouse corneal samples with an additional step of mechanical tissue disruption using MagNA Lyser beads in a MagNA Lyser Instrument (Roche). The expression fold changes were presented as mean ± SD. Significance was determined by the nonparametric Mann–Whitney U test.

Mouse eyes with and without injuries were enucleated at 2 weeks post-injury and treatment. The isolated corneas were placed in ice-cold RLT buffer (Qiagen) with 1% β-mercaptoethanol (Sigma-Aldrich) and disrupted in MagNA Lyser Green Beads (Roche, Basel, Switzerland) at 6000 rpm for 50 s for 6 cycles in a MagNA Lyser (Roche), with intermittent cooling between cycles. The lysate was passed through a Qiashredder (Qiagen), and total RNA was extracted using RNeasy Miniprep kit (Qiagen) and an on-column RNase-free DNase kit, respectively. Reverse transcription of RNA (500 ng) was done with SuperScript III Reverse Transcriptase kit (Thermo Fisher) and random primer hexanucleotides (10 ng/mL, Thermo Fisher). Target gene expression was assayed with specific primer pairs (Supplementary Table S2) using SYBR Green Real-Time Master Mix (Life Technologies, Carlsbad, CA, USA) in a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Experiments were run as technical triplicate. The relative RNA abundance was determined by ΔΔCT after normalization with the housekeeping 18S genes, and fold changes were expressed as mean ± SD. Significance was determined by a non-parametric Mann–Whitney U test.