nucleic acid hybridization to array protocol
Overall 25,262 transcripts were represented on the microarray by 1, 2 or 3 individual probes. Hybridizations for all experimental conditions were performed in 4 replicates. Total RNA samples derived from the treatments were hybridized against the pooled control respective to their origin. The microarray hybridization procedure was carried out with 300ng of cyanine-3 and cyanine-5 labelled cRNA for 17h at 65°C. Control/control hybridization were performed, each component of the pooled control (LP 2°C, LP 7°C, LP 12°C) was hybridized against the pooled control to mitigate dye bias effects. Subsequently microarray disassembly and wash procedure followed as described by the manufacturer’s instructions (Agilent).
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Agilent publication number: G4140-90050
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
Hybridization signals were recorded by Agilent G2505B scanner, and analyzed by Feature Extraction 9.3 (Agilent Technologies) and Spotfire 8.0 (Spotfire Inc., Cambridge, MA, USA) software. (Parameters: Scanning hardware = OTHER: Agilent Technologies Scanner G2505B, Scanning software = Feature Extraction Software [Agilent])
KBD patient came from the KBD prevalent areas-Linyou country and Yongshou country of Xi’an city of Shaanxi province. Based on the radiography of right hand, knee and hip joints, and cartilage sections after hematoxylin and eosin (H&E) staining, the KBD patient was diagnosed as having grade II or III KBD according to the KBD clinical diagnosis criteria of China (Diagnostic code GB16395-1996). Cartilage sample was derived from the femoral condyles of knee. The obtained cartilage sample was rapidly dissected and frozen in liquid nitrogen, and subsequently stored at -80C until RNA extraction.
Frozen cartilage of each sample was first rapidly ground in liquid nitrogen. Total RNA was then isolated from cartilage samples using the Agilent Total RNA Isolation Mini kit (Agilent Technologies, Santa Clara, CA, USA) following manufacturer recommended protocol. The quality and integrity of extracted total RNA were evaluated with 1% agarose gel electrophoresis. (Parameters: Extracted product = total_RNA, Amplification = none)
OA patient came from the non–KBD prevalent areas of Xi’an city. The OA patients were diagnosed as having primary knee OA according to the Western Ontario and McMaster Universities Osteoarthritis Index (including pain, joint stiffness, physical, social,emotional function), clinical evaluation and radiologic imaging, which allowed for exclusion of KBD, rheumatoid arthritis (RA) and genetic bone and cartilage diseases. Cartilage sample was derived from the femoral condyles of knee. The obtained cartilage sample was rapidly dissected and frozen in liquid nitrogen, and subsequently stored at -80C until RNA extraction.