Genechip fluidics 450

RNA was extracted using Trizol Reagent (Life Technologies, Inc, New York, USA) according to the manufacturer’s protocol. Total RNA was purified with RNeasy Plus Micro Kit (Qiagen, Valencia, USA) to remove genomic DNA. The RNA integrity number (RIN) was measured with Agilent RNA 6000 NanoChips on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). The quantity was measured with a NanoDrop 1000 (NanoDrop Technologies, Inc, Wilmington, USA). A total of 300 ng of each RNA sample with RIN higher than 9 was labeled with GeneChip Whole Transcript (WT) Sense Target Labeling Assay (Affymetrix, Santa Clara, USA) and hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays at 45 °C for 16 h. Arrays were stained and washed on Affymetrix GeneChip Fluidics 450 following the manufacturer’s protocol and scanned with an Affymetrix GeneChip Scanner 3000 7G System. Array data files were generated with GeneChip® Command Console® Software (AGCC) (Affymetrix, Santa Clara, USA) (ArrayExpress accession ID# E-MTAB-11492) and statistical analysis was carried out on software of GeneSpring^TM (Agilent Technologies). The fold change between normal and OA samples was based on the p < 0.05 from a t-test (Asymptotic and Benjamini Hochberg FDR).

Biotin-labelled antisense RNA (aRNA) was prepared from 100 ng of genomic DNA purified from total RNA using a GeneChip® 3’-IVT express kit (Affymetrix, Santa Clara, CA, USA). Furthermore, 12 μg of biotin-labelled aRNA was fragmented (35–200 nt) at 95 °C for 35 min for hybridization. Fragmented biotin-labelled aRNA were hybridized to an Affymetrix® GeneChip® Bovine genome Array at 45 °C for 16–18 h. Arrays were stained with streptavidin-phycoerythrin and washed using Affymetrix® GeneChip Fluidics 450 following manufacturer’s protocol and scanned with an Affymetrix® GeneChip Scanner 3000 7G System at the Southern Alberta Microarray Facility (Calgary, AB, Canada).

Total RNA was isolated from CASMC using the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. The isolated RNA was quantified by UV absorption with a NanoDrop 1000 (NanoDrop Technologies, Inc.) and the quality verified with an Agilent RNA 6000 NanoChip on a 2100 Bioanalyzer (Agilent Technologies). Samples with an RNA integrity number (RIN) ≥ 8 were considered suitable for use in microarrays. A total of 250 ng of RNA in each sample with RIN > 9 was reverse transcribed, amplified, labeled with the Ambion WT Express kit (Ambion) and hybridized to Human Gene 1.0 ST arrays (Affymetrix) at 45°C for 16 h. The arrays were washed and stained on an Affymetrix GeneChip Fluidics-450 following the manufacturer’s protocol and scanned on an Affymetrix GeneChip Scanner 3000 7G System. The raw data sets for array comparisons have been deposited in the Gene Expression Omnibus website: www.ncbi.nlm.nih.gov/geo/ (accession number: GSE56819). Of the 28,868 genes represented on the GeneChip Human Gene 1.0ST array, genes for which the log 2-transformed signal intensities differed between the negative control and ROCK1- or ZIPK-knockdown groups by more than 0.585 (1.5-fold change without adjustment) were identified as genes whose expression was altered significantly by the kinase knockdown.