Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray

Yoo, Hyun Jung; Yoon, Sung Soo; Park, Seon Yang; Lee, Eun Young; Lee, Eun Bong; Kim, Ju Han; Song, Yeong Wook

Total RNA was processed using the Genesys Applied Biosystem Facility (Genesys, Munster, Germany), according to manufacturer's recommendations. Each RNA pool (2 μg) was labeled with Digoxigenin-UTP using the ABI Chemiluminescent RT-IVT Labeling Kit v 1.0. Double-stranded cDNA was prepared from total RNA. UTP-digoxigenin-labeled complementary RNA (cRNA) was synthesized by in vitro transcription. Labeled cRNA (10 μg) was hybridized to ABI Human Genome Survey Microarray v 2.0, which was then incubated with alkaline phosphatase-linked digoxigenin antibody. Phosphatase activity was then initiated to produce the chemiluminescent signal. Chemiluminescent (cRNA) and fluorescent (spot background) signals of the cRNA and standard control spots were then scanned. Chemiluminescent detection and image acquisition was performed using an Applied Biosystems 1,700 (AB1,700) Chemiluminescent Microarrays Analyzer, according to the manufacturer's instructions. Using the software supplied with the AB1,700 apparatus, the spot chemiluminescent signal was normalized over the fluorescent signal of the same spot to obtain normalized signal value. For inter-array normalization, global median normalization was ap-plied across all microarrays (7).