Subcultures of 32 holoclones (Table 1 ) from oral mucosa, limbus and conjunctival epithelia were performed.
Analysis of holoclones’ transcriptomes was carried out using Affymetrix HG-U133 Plus 2.0 array (Thermo Fisher Scientific, Waltham, MA, USA) [20 (link)]. Keratinocytes subcultured from each holoclone were feeder-depleted using immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). According to the manufacturer’s protocol, total RNA was isolated with the Invitrogen™ PureLink™ RNA Micro Scale Kit (Thermo Fisher Scientific, Waltham, MA, USA). Differentially expressed genes (DEGs) were identified on robust multiarray average (RMA)-normalized data through the ANOVA module supplied by the Partek GS. 6.6 Software Package (ver. 7.21.1119, Chesterfield, MO, USA). The probesets displaying a fold change contrast ≥ 2 and a false discovery rate (FDR) < 0.05 were selected as DEGs among oral mucosa, limbal and conjunctival holoclones. Integral gene expression data are deposited to the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo ; series GSE198408). The network of angiogenesis-related transcripts was generated using QIAGEN IPA® (ver. 8.6, QIAGEN Inc., Hilden, Germany, https://digitalinsights.qiagen.com/IPA ) [80 (link)].
Analysis of holoclones’ transcriptomes was carried out using Affymetrix HG-U133 Plus 2.0 array (Thermo Fisher Scientific, Waltham, MA, USA) [20 (link)]. Keratinocytes subcultured from each holoclone were feeder-depleted using immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). According to the manufacturer’s protocol, total RNA was isolated with the Invitrogen™ PureLink™ RNA Micro Scale Kit (Thermo Fisher Scientific, Waltham, MA, USA). Differentially expressed genes (DEGs) were identified on robust multiarray average (RMA)-normalized data through the ANOVA module supplied by the Partek GS. 6.6 Software Package (ver. 7.21.1119, Chesterfield, MO, USA). The probesets displaying a fold change contrast ≥ 2 and a false discovery rate (FDR) < 0.05 were selected as DEGs among oral mucosa, limbal and conjunctival holoclones. Integral gene expression data are deposited to the Gene Expression Omnibus repository (
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