ID_REF = VALUE = PLIER value
Arrays were scanned using GeneChip Scanner 3000 7G and Command Console Software v. 3.2.3 to produce .CEL intensity files.
RNA were extracted from the plated cells with the Purelink RNA miniprep kit (Ambion) 72 hours after transfection.
The probe cell intensity files (*.CEL) were analyzed in Affymetrix Expression Console software v1.1.1 using the PLIER algorithm to generate probe level summarization files. The settings used were algorithm-PLIER v2.0; quantification scale-Linear; quantification type-signal and detection p-value; background-PM-GCBG; normalization method-sketch-quantile.
P3 HCEP cells were trypsinized and plated 100,000 cells/well into a 12-well dish containing 1ul lipofectamine RNAiMAX and 30nM EHF or scramble siRNA pre-mixed 20 prior to cell plating. The EHF siRNA were pooled from 3 individual siRNA targeting EHF (Ambion, ID s25397, s25398, s25399) in the same concentration.
2ug of the labeled, fragmented single-stranded cDNA is hybridized at 45oC with rotation for 17 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix GeneChip 1.0 ST array. The GeneChip arrays were washed and then stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 450 (Fluidics protocol FS450_007).
The Ambion WT expression kit (Life Technologies, Carlsbad, CA) was used to prepare RNA samples for whole transcriptome microarray analysis. Briefly, random hexamers that are tagged with a T7 promoter are used in first strand synthesis of cDNA. Then, using the T7 promoter, second strand synthesis is performed and the double-stranded cDNA is subsequently used as a template in an in vitro transcription reaction to generate many copies of antisense cRNA. 10ug of antisense cRNA is input into a second cycle cDNA reaction using reverse transcriptase and random hexamers to produce single-stranded DNA in the sense orientation. The single-stranded DNA is fragmented to an average length of 70 bases and then labeled using a recombinant terminal deoxynucleotidyl transferase (TdT) and an Affymetrix proprietary DNA labeling reagent that is covalently linked to biotin.
Primary human corneal epithelial cells (HCEPs) from CELLnTEC Inc. were grown in CnT-20 medium as directed by the manufacturer.
Corneas from wildtype CB6 mice were collected at the respective ages.
Affymetrix GeneChip 3000 Scanner 7G.
Using the NuGEN Ovation RNA Amplification System V2 (NuGEN Technologies, San Carlos, CA), first strand cDNA was synthesized from the poly(A)+ mRNA present within the isolated total RNA (20 ng total RNA starting material used in each sample reaction). Fragmentation of the mRNA within the resulting cDNA/mRNA complexes created priming sites for DNA polymerase to synthesize DNA complementary to the first strand cDNAs. The resulting ds cDNAs have a unique DNA/RNA heteroduplex at one end. These ds DNA templates were used to perform linear isothermal DNA amplification (SPIA, NuGEN Technologies, San Carlos, CA). 3.75 μg of the resulting amplified cDNA material for each sample was first fragmented and subsequently end-labeled using the NuGEN FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies, San Carlos CA).
The results were quantified and analyzed using Expression Console ver.1.1 software (Affymetrix, Inc.) using the PLIER Algorithm default values (Quantification Scale: Linear; Quantification Type: Signal and Detection P-Value; Background: PM-GCBG; Normalization Method: Sketch-Quantile).
2.2 μg of the resulting fragmented, biotin-tagged cDNA was placed into a hybridization cocktail (220 μL), with 200 μL actually hybridized at 45°C with rotation for 18 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix Mouse Gene 1.0 ST array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450 using the FS450_0004 fluidics protocol.
Whole eye globes were removed and corneas dissected, removing all surrounding tissues. Corneal epithelium was isolated by incubation of total cornea in dispase II for 2h at 37C. Total RNA was extracted with Trizol followed by clean-up with the RNeasy micro kit. Corneal stroma was isolated by embedding eye globes in OCT, sectioning at 8 um, and performing laser capture microdissection. Total RNA was isolated with the RNeasy micro kit.