Mirna sequence specific primers

QRT-PCR was performed as described previously^{14 (link)}. Briefly, 10 ng of total RNA were reverse transcribed (RT) using Taqman MicroRNA RT kits and miRNA sequence-specific primers (Thermo Fisher Scientific). QPCR was carried out using Taqman 2X universal PCR Master Mix (no AmpErase UNG) along with Taqman 20X MicroRNA Assays (Thermo Fisher Scientific). Each well contained 1.33 µl of RT reaction product, 1X Taqman PCR Master Mix, and 1X specific miRNA primer, designed to detect mature miRNAs. Amplification was carried out on the ViiA^TM 7 Real Time PCR System (Thermo Fisher Scientific). Each sample was run in triplicate. Signals were normalized to small nucleolar RNA U75 (SNORDU75). U75 housekeeping miRNA run in parallel. A comparative threshold cycle (Ct) method (ΔΔCt) was used to calculate relative miRNA expression. High-throughput QRT-PCR on a custom gene panel was performed on a Fluidigm BioMark HD system in the Genomic Core at Cedars-Sinai Medical Center per manufacture instruction. Primers for miR-10b predicted target and putative LESC and corneal epithelial marker genes are listed in Supplementary Table S6. In situ hybridization was performed as described previously12 (link).

QPCR was performed as described previously [25] (link). Briefly, 10 ng of total RNA were reverse transcribed (RT) using Taqman MicroRNA RT kits and miRNA sequence-specific primers (Thermo Fisher Scientific). QPCR was carried out in MicroAmp Optical 384-well plates using Taqman 2X universal PCR Master Mix (no AmpErase UNG) along with Taqman 20X MicroRNA Assays (Thermo Fisher Scientific). Each well contained 1.33 µl of RT reaction product, 1X Taqman PCR Master Mix, and 1X specific miRNA primer, designed to detect mature miRNAs. Amplification was carried out on the ViiA 7 Real Time PCR System (Thermo Fisher Scientific). Each sample was run in triplicate. Signals were normalized to the U75 housekeeping miRNA run in parallel. A comparative threshold cycle (Ct) method (ΔΔCt) was used to calculate relative miRNA expression.