developmental stage: adult - 15 years tissue: cartilage tissue subtype: deep (AACD) adult articular cartilage zone
Treatment protocol
Cartilage samples and the E40 hindlimbs were placed in the cryostat chamber for temperature equilibration, embedded in frozen section medium (Neg-50,ThermoFisher, Walldorf) and sectioned along their sagittal axis using a cryostat (Hyrax C 50 Cryostat, Carl Zeiss Microscopy GmbH, Jena, Germany) at a −25°C chamber/chuck temperature. Sections (10μm) were mounted onto precooled RNAse-free polyethylene naphthalate (PEN)-coated slides (Zeiss MembraneSlide 1.0 PEN NF) and stained with cresyl violet according to a standard protocol from Zeiss Labs, Munich, Germany (step 1: fixation in 70% Ethanol for 2 min; step 2: 30 sec in 1% cresyl violet acetate solution; step 3: dip into 70% followed by 100% Ethanol; step 4: air-dry 1-2 min). The cartilage thickness was measured from the articular surface to the tidemark, confirming the reported cartilage thickness of the equine stifle of 1500 – 2000 μm [30] and divided into a superficial (superficial 10%; AACS), middle (adjacent 20-30%; AACM) and deep (50-90% depth; AACD) layer (figure 1) [3,14]. Cells were selected from the ACCS, ACCM, ACCD, II, OI and EC using laser capture microdissection (PALM MicroBeam system, Carl Zeiss Microscopy GmbH), catapulting the selected tissues of each sample group directly into separate 500 μl AdhesiveCap tubes (Carl Zeiss Microscopy GmbH), avoiding contact with any surface and thereby RNase contamination.
Extracted molecule
total RNA
Extraction protocol
The harvested cells were lysed for 30 minutes using RLT lysis buffer (RNeasy® Micro kit, Qiagen Sciences Inc, Hilden, Germany), then vortexed and frozen at −20°C. Total RNA was extracted from cell lysates using RNeasy Micro Kits according to manufacturer's protocols. RNA was stored at −80°C.
Label
Cy3
Label protocol
RNA was amplified using SuperAmp (Agilent Technologies, Paolo Alto, CA, USA), a global PCR protocol using mRNA-derived cDNA, according to the manufacturer’s protocol. Yields of cDNA were measured with a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). The integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform. 250 ng of each of the cDNAs was labelled with Cyanine 3-CTP (Cy-3)
Hybridization protocol
samples were hybridized overnight (17 hours, 65°C) to an Agilent Whole Horse Genome Oligo Microarrays 8x60K using Agilent’s recommended hybridization chamber and oven.
Scan protocol
Fluorescence signals of the hybridized microarrays were detected using Agilent’s Microarray Scanner System.
Description
gene expression articular cartilage zones
Data processing
The Agilent Feature Extraction Software was used to read out and process the microarray image files using default parameters.
