Cell line Growth Protocol Other: MCF10A (benign mammary epithelial), LNCAP (prostate carcinoma), Jurkat (T-cell lymphoma), SUDHL6 (germinal center B-cell like diffuse large B-cell lymphoma), OCI-Ly6 (activated B-cell like diffuse rare B-cell lymphoma) and L428 (Hodgkin's lymphoma) were grown under standard conditions (Chen et al., 1996)
Extracted molecule
total RNA
Extraction protocol
RNA Extraction Protocol Other: RNA was isolated from the cells using TriReagent following the manufacturer's protocol (Molecular Research Center, Inc., Cincinnati, OH). The integrity of the RNA was confirmed by analysis with the Agilent 2100 Bioanalyzer (Palo Alto, CA) using the RNA6000LabChipkit.
Label
cy3
Label protocol
Cy3 Labeling Protocol Label amount: 20 ug Label method: indirect amino-allyl method Other: The cDNA was synthesized and labeled by the indirect amino-allyl method using reagents and protocols supplied with the Strategene FairPlay(TM) Microarray Labeling Kit. For cDNA synthesis, Stratascript reagents (Stratagene) were used, and Cy3 fluorophore amino-allyl reagents were obtained from Amersham (Piscataway, NJ). For each synthesis, 20 ug of total RNA were used. Labeled cDNA targets were purified using the Minelute purification kits (Qiagen, Valencia, CA).
Cell type: mixture of liver, testis, mammary gland, cervix, brain, skin, liposarcoma, macrophage, T-lymphoblast and B-lymphocyte
Biomaterial provider
Stratagene (La Jolla, CA)
Extracted molecule
total RNA
Label
cy5
Label protocol
Cy5 Labeling Protocol Label amount: 20 ug Label method: indirect amino-allyl method Other: The cDNA was synthesized and labeled by the indirect amino-allyl method using reagents and protocols supplied with the Strategene FairPlay(TM) Microarray Labeling Kit. For cDNA synthesis, Stratascript reagents (Stratagene) were used, and Cy5 fluorophore amino-allyl reagents were obtained from Amersham (Piscataway, NJ). For each synthesis, 20 ug of total RNA were used. Labeled cDNA targets were purified using the Minelute purification kits (Qiagen, Valencia, CA).
Hybridization protocol
Oligo Hybridization Protocol Other: The microarrays were prehybridized in 40 ul of 5X SSC, 0.1% SDS and 1% BSA at 42C for 30 min. The prehybridization solution was removed and arrays were hybridized for 16 h at 42C in 5X SSC buffer containing Cy3/Cy5 labeled targets, 25% formamide, 0.1% SDS, 1 ug Cot-1 DNA and 1 ug polyA RNA. The arrays were washed at room temperature in 2x SSC, 0.1% SDS for 2 min, 1X SSC for 2 min, and 0.2X SSC for 2 min. The slides were dried by spinning at 650 r.p.m. for 3 min.
Description
mAdb experiment ID: 45655
Data processing
Data were background corrected by subtracting the median background pixel intensity from the mean foreground intensity, because the median background subtraction makes the tiny dust particles less significant and the mean foreground is preferable to the median foreground for spots that lack signal in the center, called doughnuts. Signals < 100 were floored at 100. Spots flagged for poor quality were eliminated from the analysis and genes with missing data were eliminated.
