|
| Status |
Public on Sep 08, 2009 |
| Title |
HOB replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
normal human osteoblasts
|
| Organism |
Homo sapiens |
| Characteristics |
Normal Human Osteoblast Cells from healthy male donor (Heidelberg, Germany, Catalogue # C-12760)
|
| Treatment protocol |
Cells were harvested with Tripsin (Invitrogen) as per manifacturers instructions
|
| Growth protocol |
The human OS cell lines U2OS (ATCC # HTB-96) and MG63 (ATCC # CRL-1427) were purchased from American Type Culture Collection ATCC (Rockville, MD) and maintained in alpha-Minimum Essential Medium (alpha-MEM) supplemented with 10% heat inactivated Fetal Bovine Serum and 2 mM L-Glutamine. Normal human osteoblasts (HOB) are primary osteoblasts from the hip bone of a normal male donor that were purchased from PromoCell (Heidelberg, Germany, Catalogue # C-12760) and maintained in medium provided by the manufacturer and used at culture passage 3. Three days after plating (~80% confluent), cells where harvested for DNA or RNA extractions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from U2OS, MG63 cells and HOB was extracted using the TRIzol reagent method. Cells were washed 3 times by Phosphate Buffer Saline (PBS) and lysed in the culture vessel using TRizol (Invitrogen, Osaka, Japan). 1 ml of TRIzol was used for every 100 mm culture plates an RNase free environment. Chloroform was then added (200 µl for each 1 ml TRIzol) and the samples were centrifuged at high speed for 15 minutes at 4C. The aqueous layer was then transferred into a new tube and RNA was precipitated with iso-propanol followed by one wash using 70% ethanol. The RNA precipitate was then dissolved in 10-15 µl of RNAse free water
|
| Label |
TdT labeling kit
|
| Label protocol |
Labeling, hybridization and scanning were performed as per Affymetrix GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual version 4 (P/N 701880 Rev 4). (http://www.affymetrix.com/support/technical/byproduct.affx?product=hugene-1_0-st-v1).
|
| |
|
| Hybridization protocol |
Labeling, hybridization and scanning were performed as per Affymetrix GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual version 4 (P/N 701880 Rev 4). (http://www.affymetrix.com/support/technical/byproduct.affx?product=hugene-1_0-st-v1).
|
| Scan protocol |
Labeling, hybridization and scanning were performed as per Affymetrix GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual version 4 (P/N 701880 Rev 4). (http://www.affymetrix.com/support/technical/byproduct.affx?product=hugene-1_0-st-v1).
|
| Description |
gene expression data from HOB bioreplicate 2
|
| Data processing |
Data from RNA expression array experiments in the form of .cel files (GCOS 1.3 software) were imported into and analysed using the Partek Genomic Suite Software (PGS). The expression array .cel files for HOB, U2OS, and MG63 (2 arrays each) were imported using PGS Gene Expression Workflow tool (subject to RMA normalization and log2 transformation). The significantly over- and under-expressed genes in U2OS and in MG63 cell lines compared to HOB were detected using 1-way ANOVA tool at p<0.01 and +/- 2-fold enrichment. Significantly over- and under-expressed regions were annotated to the corresponding genes present on the Affymetrix Gene 1.0 Array using the HuGene-1_0-st-v1.na24.hg18.transcript.csv file.
|
| |
|
| Submission date |
May 12, 2008 |
| Last update date |
Sep 08, 2009 |
| Contact name |
Bekim Sadikovic |
| E-mail(s) |
[email protected]
|
| Organization name |
The SickKids Hospital and Princess Margaret Hospital
|
| Street address |
555 University Avenue
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1X8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6244 |
| Series (4)
|
| GSE11414 |
Gene expression of osteosarcoma cell (U2OS, MG63) lines relative to normal human osteoblasts (HOB) |
| GSE11416 |
Comparison of osteosarcoma cell lines and normal human osteoblasts |
| GSE12865 |
Gene expression of human paediatric osteosarcoma tumour samples relative to normal human osteoblasts |
| GSE12885 |
Genome-wide changes in DNA methylation and copy number play a role in deregulation of gene expression in osteosarcoma |
|
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