cell type: human dermal firbroblasts (hDFs) phenotype: normal
Extracted molecule
total RNA
Extraction protocol
cell isolation protocol: Normal aortic and mitral valves were obtained from five healthy donor hearts following the donors’deaths in traffic accidents (three males and two females, 25 to 34 years old). Small portions of each valve were used for VIC isolation. Human aortic interstitial cells (hAVICs) and mitral valve interstitial cells (hMVICs) were isolated by collagenase digestion as previously described [Taylor et al.J Heart Valve Dis 2000;9:150-158.] and then cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% penicillin-streptomycin, and 2 mM L-glutamine.Primary Cells were used for microarray analysis. Total RNA was extracted using Trizol reagent (Invitrogen) and further purified using NucleoSpin® RNA clean-up (MACHEREY-NAGEL, Germany) according to the manufacturer’s instructions. The RNA quality was assessed with formaldehyde agarose gel electrophoresis and quantitated spectrophotometrically. Equivalent RNA from primary VICs of five donors was pooled, and 1ug total RNA was converted to cDNA and labeled for advance microarray according to the manufacturer’s protocol (CapitalBio Corporation, Beijing, China).
Label
Cy3
Label protocol
cDNA labeled with a fluorescent dye (Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. This procedure has been previously described[Guo,Y. et al. J. Virol. 2005,79:14392-14403.], and the procedure has been improved by using CapitalBio cRNA Amplification and Labeling Kit (CapitalBio) for producing higher yields of labeled cDNA.
Hybridization protocol
Hybridization was carried out according to the NimbleGen`s Expression user`s guide(see manufacture`s user guide)and performed at CapitalBio Corporation(Beijing,China). See www.nimblegen.com.
Scan protocol
Scanning was carried out according to the NimbleGen`s Expression user`s guide(see manufacture`s user guide)and performed at CapitalBio Corporation(Beijing,China). See www.nimblegen.com.
Description
HDF
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
