Target Source: human bone marrow stromal cells, HS-27a Description: human bone marrow stromal cells cultured in RPMI medium 1640 containing 10% fetal calf serum. Values are ratio of HS-27a to Universal RNA.
HS-27a cells were plated in T75 flasks at 3,000,000 cells/flask. They were cultured for 3 days and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. As a control RNA, Universal Human Reference RNA (Stratagene, La Jolla, CA) which is a mixture of RNA from 10 human cell lines approximating the expression profile of the majority of human genes was used. The RNA (30 µg) was annealed with 5 µg oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42°C in the presence of 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 0.2 M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS-27a RNA and Human Universal RNA was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 µl of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data table.
