Invitrogen purelink rna micro scale kit

Transcriptome analysis was performed by using the Affymetrix HG-133 plus 2.0 array. 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 a robust multiarray average (RMA)-normalized data through the ANOVA module supplied by the Partek GS. 6.6 Software Package [49 (link)]. 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 were submitted to the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo; series GSE198408). Real-time RT-PCR was performed to validate microarray data as described within the Supplementary Material.

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)].