Cultured FLS were cultured to ~80% confluency, and then starved in DMEM containing 1% FCS for 24 hours. The cells were placed in culture medium containing 1% FCS in the presence or absence of TNF for 24 hours. Total RNA was extracted with Ambion RNAqueous Kit (Ambion, Austin, TX) according to manufacturer’s protocol.
Label
Cy3 and Cy5
Label protocol
Total RNA was used for fluorescently labeled cRNA synthesis using Agilent Low RNA input Fluor Linear Amp Kit and protocol (Agilent, Santa Clara, CA).
Hybridization protocol
For hybridization, Cy3- or Cy5-labeled cRNA from treated and vehicle treated cells were combined and hybridized to an Agilent 44K Whole Rat Genome Oligo Microarray (Agilent, Santa Clara, CA) according to manufacturer’s protocol.
Scan protocol
The oligonucleotide microarray slides were scanned with Agilent microarray scanner, and the signal intensity normalization was done with proprietary software from Agilent.
