Spike in kit

Total RNA was purified from cell lines using the miRNeasy kit (Qiagen, Valencia, CA, USA), and 100 ng was labeled and hybridized to the array using the miRNA complete labeling and hyb kit (Agilent, Santa Clara, CA, USA) and spike-in kit (Agilent). Data were generated on a GeneChip Scanner 3000 (Agilent), Human microRNA Microarray Release 16.0, 8x60K arrays (Agilent). Data were imported into R, RMA normalized using the package AgiMicroRna,^{46 (link)} summarized to the log2 scale and returned for further association analysis as a gene by sample matrix. The miR expression data are MIAME compliant and have been submitted to the Gene Expression Omnibus (GSE73774). Differences in microRNA expression were determined with Omics Explorer (Qlucore, Lund, Sweden), using ANOVA on 549 miRs from TCGA^{12 (link)} and 1368 miRs from cell lines using using a cutoff of P<0.05 and false discovery rate of 0.09 and 0.73, respectively, calculated using the Benjamini–Hochberg method. Fold change of miRs found to be significant (P<0.05) in both TCGA and cell lines was then determined by comparing expression in each cluster with the other two clusters (Table 3).

Agilent Technologies SurePrint Gene Expression Microarrays (Agilent Techologies, Santa Clara, CA, USA) were used to profile gene expression of RNA samples. Samples were processed according to Agilent Two-color Microarray Based Gene Expression Analysis (Low Input Quick Amp Labeling Kit protocol) by Agilent Spike-In Kit, according to the manufacturer’s instructions. The Agilent Two-Color Microarray-based Gene Expression Analysis uses Cyanine 3- and Cyanine 5-labeled targets to measure gene expression in experimental and control samples. Equal amounts of Cyanine 3-labeled (control samples) and Cyanine 5-labeled (experimental samples) cRNA from samples were simultaneously co-hybridized onto the arrayed oligonucleotides on the same Agilent 44k Whole Human Genome chip (Agilent Technologies, 4 × 44k format) slide at 65°C for 17 h using an Agilent Gene Expression Hybridization Kit in Agilent’s SureHyb Hybridization Chambers (G2545A). The hybridized microarrays were washed according to manufacturer’s instructions, and then scanned by Agilent Feature Extraction software (10.5, Protocol GE2_105_Dec08).

Normal human dermal fibroblasts were seeded into six-well microplates at 40,000 cells/well. NHDF-F22Br (female 22y breast skin), M50F (male 50y face skin), F60Br (female 60y breast skin), and PromoCell NHDF-c64a were cultured in complete DMEM for 4 days. NHDF were equilibrated in low (1%) serum DMEM for 24 h and treated with or without transforming growth factor-β1 (TGF-β1) (10 ng/ml) in low serum DMEM for a further 12 or 24 h. Cells were lysed and RNA was extracted using the RNeasy mini kit protocol. RNA concentration and integrity were measured using a Bioanalyser 2100 (Agilent) and RNA 6000 Nano kit (Agilent) according to the manufacturer’s instructions. The one-colour Quick-Amp labelling kit (Agilent) was used to derive cyanine 3-cytidine triphosphate (Cy3-CTP) labelled cRNA, and the spike-in kit (Agilent) was used to monitor sample amplification and labelling efficiency. RNA was converted to cDNA using the cDNA mix kit (Quick-Amp). See Supplementary Materials and Methods for hybridisation and analysis protocols.