|
| Status |
Public on Dec 14, 2007 |
| Title |
PARVB/3D/bovine type I collagen |
| Sample type |
RNA |
| |
|
| Source name |
MDA-MB-231_PARVB cells (PARVB long isoform)
|
| Organism |
Homo sapiens |
| Characteristics |
MDA-MB-231 human metastatic breast ductal carcinoma cell line derived from pleural effusion transfected with human Parvin-beta (long isoform) cloned into the pcDNA3.1 Myc-His (clone B) plasmid and selected for stable integration using G418.
|
| Biomaterial provider |
Hospital for Sick Children, Toronto, Canada
|
| Treatment protocol |
MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37oC in a 5% carbon dioxide-containing incubator without antibiotics or anti-fungal agents.
|
| Growth protocol |
For embedded growth in type I collagen, cells (3 x 105) were mixed with 1.5mg/ml bovine epidermal type I collagen (PureCol, Inamed Biomaterials, Fremont, CA) to a total volume of 2ml, and then added to 6 well plates. The collagen was allowed to set at 37oC prior to addition of 10% FBS-containing DMEM culture medium. Cells were cultured for 7 days with a change of medium every 2 days.
and allowed to set prior to addition of 10% FBS-supplemented DME medium. Cells were cultured for 7 days with a change of medium every 2 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10ml of Trizol reagent (Invitrogen) was added to the well containing MDA-MB-231_PARVB3 cells embedded in bovine type I collagen. The cells and collagen (approximately 2ml volume) were digested in Trizol by drawing the mixture through a 21G needle (at least 10 times) times followed by incubation at room temperature for 10-15min in a 15ml sterile plastic tube. 2.5ml of chloroform was then added and the mixture shaken vigorously. The sample was then centrifuged at 11,000rpm (at 4oC) for 30min and the clear upper (aqueous) layer transferred to a sterile 15ml glass tube. 5ml of isopropanol was then added and the mixture cooled at -20oC for at least 20min. The mixture was then centrifuged at 11,000rpm (4oC) for 30min and the RNA pellet washed thoroughly with 75% ethanol in DEPC-treated sterile water. RNA was then resuspended in DEPC-treated sterile water.
|
| Label |
biotinylated CTP and UTP
|
| Label protocol |
All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. 8ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase (Invitrogen) primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript. The resulting cRNA was used as template for random-primed cDNA synthesis and a second round of in vitro transcription, which incorporated biotinylated CTP and UTP.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Affymetrix U133_Plus_2 gene chip microarrays. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
| Description |
The MAS5 algorithm was used to determine gene expression ratios [Flagged as I (increased), D (decreased), MI (marginally increased), MD (marginally decreased), or NC (no change)], and p values for the change. Ratios of mRNA expression level between 231_VC and 231_PARVb cells were calculated by determining the inverse log2 of the signal log ratio (S.L.R.) for each differentially-expressed gene. Array results from 2D and 3D experiments were compared, and Venn diagrams generated using Genespring GX software (Agilent Technologies, Palo Alto, CA).
|
| Data processing |
Affymetrix Microarray Suite 5.0 was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. A weighted mean of probe fluorescence (corrected for nonspecific signal by subtracting the mismatch probe value) was calculated using the One-step Tukey's Biweight Estimate. This Signal value, a relative measure of the expression level, was computed for each assayed gene. Global scaling was applied to allow comparison of gene signals across multiple microarrays: after exclusion of the highest and lowest 2%, the average total chip signal was calculated and used to determine what scaling factor was required to adjust the chip average to an arbitrary target of 150. All signal values from one microarray were then multiplied by the appropriate scaling factor.
|
| |
|
| Submission date |
Dec 02, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Cameron N Johnstone |
| E-mail(s) |
cameron.johnstone@petermac.org
|
| Organization name |
Peter MacCallum Cancer Centre
|
| Department |
Research Division
|
| Lab |
Cancer Metastasis Laboratory
|
| Street address |
2 St. Andrew's Place
|
| City |
East Melbourne |
| State/province |
Vic |
| ZIP/Postal code |
3002 |
| Country |
Australia |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE9747 |
Regulation of gene expression in MDAMB231 breast cancer cells by Parvin-beta in 2D vs 3D culture conditions |
|
| Relations |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE119087 |
