GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Series GSE2174

Query DataSets for GSE2174

Status

Public on Jan 26, 2005

Title

D4-ME effects

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

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

Contributor(s)

Madlambayan GJ, Kirouac DC, Zandstra PW

Citation missing

Has this study been published? Please login to update or notify GEO.

Submission date

Jan 21, 2005

Last update date

Aug 10, 2018

Contact name

Daniel Kirouac

E-mail(s)

daniel.kirouac@utoronto.ca