Total RNA from fibroblats was prepared using RNAeasy kit (Qiagen, Valencia, CA).
Label
cy3
Label protocol
cDNA samples generated from RNA samples and purified before the coupling reaction were labeled with Cy3 (for fibroblast samples) or Cy5 (for reference RNA) mono-reactive dyes (GE Healthcare, Piscataway, NJ), and were hybridized simultaneously on an oligo-DNA chip (Hs-OperonV3.0-v1p24, p27, p31) overnight at 42°C.
reference: Equal quantities of total RNA from each cell line (brain, breast, B-lymphocyte, cervix, liver, liposarcoma, macrophages, skin, testis, Y-lymphocyte) were pooled together. Provided by Stratagene (La Jolla, CA).
Extracted molecule
total RNA
Extraction protocol
Total RNA from fibroblats was prepared using RNAeasy kit (Qiagen, Valencia, CA).
Label
cy5
Label protocol
cDNA samples generated from RNA samples and purified before the coupling reaction were labeled with Cy3 (for fibroblast samples) or Cy5 (for reference RNA) mono-reactive dyes (GE Healthcare, Piscataway, NJ), and were hybridized simultaneously on an oligo-DNA chip (Hs-OperonV3.0-v1p24, p27, p31) overnight at 42°C.
Hybridization protocol
Oligo-cDNA microarray hybridization was performed according to the National Cancer Institute in-house protocol. A universal human reference RNA (Stratagene, La Jolla, CA) was used as a control. cDNA samples generated from RNA samples and purified before the coupling reaction were labeled with Cy3 (for fibroblast samples) or Cy5 (for reference RNA) mono-reactive dyes (GE Healthcare, Piscataway, NJ), and were hybridized simultaneously on an oligo-DNA chip (Hs-OperonV3.0-v1p24, p27, p31) overnight at 42°C.
Scan protocol
Scanned on GenePix 4000B. Images were quantified using GenePix Pro (version 5.0.1.24).
Data processing
The data were processed by filtered 11,771 out of 36,288 spots with at least half of the corresponding intensity measures (CIMs) less than the mean of the background signal of the array plus three times the standard deviation of the background for further analysis. Data were then processed by subtracting background signals, conducting loess within-array and quantile between-array on the common reference channel normalization. One spot, Block 45, Column 18, Row 14, an empty well but passing the filtering criteria, was excluded from the data matrix.
