D4-ME effects

Umbilical cord blood-derived mononuclear cells were lineage depleted using a StemSep column and cultured in serum-free liquid suspension culture with cytokines SCF, FL, and Tpo. After four days in culture (d4), cultured cells were split into 2 groups; one subjected to media exchange and one subjected to a mock exchange, and cultured for an additional day. RNA was extracted and analyzed on an oligonucleotide microarray from day 4 cultured cells (d4), day 4-derived sorted lin+ cells (lin+), day 4 cells which were placed in fresh media for 24-hours (d5-E) and day 4 cells which were subjected to a mock media dilution/exchange and left for 24-hours (d5-NE) Total cellular mRNA was collected by lysing cells in 500 μl Trizol Reagent (Invitrogen, Groningen, The Netherlands) for 5 min followed by addition of 100 μl chloroform. After centrifugation at 12,000 g for 15 min, the aqueous phase was collected and mixed with isopropanol to precipitate the mRNA. The mRNA pellet was then obtained by centrifugation at 12,000 g for 10 min, washed in 70% ethanol and re-suspended in sterile water. mRNA concentration was then determined using a mQuant plate reader (Biotek Instruments, Winooski, VT). Subsequent sample preparation and hybridization was conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Because of the limited numbers of cells that could be collected from each experiment, the Two-Cycle Target Labeling technique was used which first translates RNA into cDNA using a T7-oligo(dT) polymerase primer. The cDNA was then subjected to in vitro transcription during which biotin labeled nucleotides are incorporated into the resultant cRNA. The cRNA was then fragmented and hybridized to the Affymetrix HU133 chip set. All samples were obtained in triplicate and sent to the Ontario Genomics Innovation Centre (Ottawa Health Research Institute, Ottawa, Canada) for analysis. Keywords: parallel sample