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Sample GSM232275 Query DataSets for GSM232275
Status Public on May 25, 2008
Title YAMC_polysomal_Rep3
Sample type other
 
Source name YAMC cells, polysomal RNA
Organism Mus musculus
Characteristics Refer to Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P. & Jat, P. S. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci U S A 90, 587-91 (1993) and Xia, M. & Land, H. Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility. Nat Struct Mol Biol 14, 215-23 (2007).
Treatment protocol RNA was harvested from ten replicates for each cell population grown in non-permissive conditions for 48 hr, followed by 24 hr in media with 0% FBS to maximize the contribution of oncogenic signaling to gene expression. RNA was collected while cells were sub-confluent and all cell populations were actively cycling.
Growth protocol Four polyclonal cell populations, control (Bleo/Neo), mp53 (p53175H/Neo), Ras (Bleo/RasV12) and mp53/Ras (p53175H/RasV12) were derived by retroviral infection of low-passage polyclonal young adult mouse colon (YAMC) cells. YAMC cells (a gift from R. Whitehead and A.W. Burgess) derived from the Immorto-mouse (aka H-2Kb/tsA58 transgenic mouse) expressing temperaturesensitive simian virus 40 large T (tsA58) under the control of an interferon γ-inducible promoter were maintained at the permissive temperature (33°C) for large T in the presence of interferon γ to support conditional immortalization in vitro. This permits expansion of the cells in tissue culture. In contrast, exposure of YAMC cells to the non-permissive temperature for large T (39°C) in the absence of interferon γ leads to growth arrest followed by cell death, indicating the absence of spontaneous immortalizing mutations in the cell population. The cells were cultured on Collagen IV-coated dishes (1μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640 medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone), 1×ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and 5 U/ml interferon γ (R&D Systems). All experiments testing the effects of RasV12 and p53175H were carried out at the non-permissive temperature for large T function (39°C) and in the absence of interferon γ.
Extracted molecule other
Extraction protocol Polysomal RNA was harvested from YAMC, bleo/neo, mp53/neo, bleo/Ras and mp53/Ras cells to obtain gene expression profiles reflective of protein synthesis rates. Cells were lysed in Extraction Buffer (50 mM MOPS, 15 mM MgCl, 150 mM NaCl, 0.5% Triton X-100 with 100 ug/mL cycloheximide, 1 mg/mL heparin, 200U RNAsin (2 uL/mL of buffer), 2mM PMSF). Supernatants were applied to 10-50% sucrose gradients, centrifuged at 36,000 rpm for 2 hr at 4°C and fractions were collected using an ISCO gradient fractionator reading absorbance at 254 nm. Polysome containing fractions were pooled and RNA was purified using the RNeasy Mini Kit (Qiagen) following the standard protocol for animal cells, except that sucrose fractions were mixed with 3.5 volumes Buffer RLT before binding to the RNeasy column. RNA was on-column DNase digested as part of the RNeasy RNA extraction protocol.
Label biotin
Label protocol Five micrograms of RNA was reverse transcribed and labeled using the mAMP kit (Ambion), with the 1x amplification protocol.
 
Hybridization protocol The cRNA yield was fragmented and hybridization cocktails were prepared using Affymetrix standard protocol for eukaryotic target hybridization. Targets were hybridized to Affymetrix Mouse Genome 430 2.0 Expression Arrays at 45°C for 16 hours, washed and stained using Affymetrix Fluidics protocol EukGE-WS2v4_450 in the Fluidics Station 450.
Scan protocol Arrays were scanned with the Affymetrix GeneChip Scanner 3000.
Description Refer to submitted paper, McMurray et al.
Data processing Expression values from the 50 microarrays processed were obtained using the RMA procedure with background correction in Bioconductor (http://www.bioconductor.org).
 
Submission date Sep 28, 2007
Last update date Aug 28, 2018
Contact name Hartmut Land
E-mail(s) land@urmc.rochester.edu
Organization name University of Rochester Medical Center
Department Biomedical Genetics
Street address 601 Elmwood Avenue, Box 633
City Rochester
State/province NY
ZIP/Postal code 14642
Country USA
 
Platform ID GPL1261
Series (1)
GSE9199 Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE RMA-processed intensity value
RAW_VALUE Background corrected, non-normalized intensity value

Data table
ID_REF VALUE RAW_VALUE
1415670_at 1304.086656 1105.016145
1415671_at 4368.352589 3725.882432
1415672_at 2991.599362 2521.075933
1415673_at 1780.382438 1510.156482
1415674_a_at 756.0311433 652.531881
1415675_at 609.3310467 529.1521147
1415676_a_at 3685.118596 3155.05426
1415677_at 579.1423758 501.1594396
1415678_at 2971.977885 2501.596588
1415679_at 1833.544343 1554.360189
1415680_at 734.9091481 635.4822041
1415681_at 2141.452004 1806.282419
1415682_at 324.0238266 285.4351734
1415683_at 3537.277183 3041.251577
1415684_at 880.205517 757.5410115
1415685_at 445.1628355 387.1908475
1415686_at 1765.575154 1497.040007
1415687_a_at 7126.882948 6287.20163
1415688_at 1201.033556 1030.075933
1415689_s_at 516.2062223 447.6577294

Total number of rows: 45101

Table truncated, full table size 1549 Kbytes.




Supplementary file Size Download File type/resource
GSM232275.CEL.gz 6.3 Mb (ftp)(http) CEL
Processed data included within Sample table

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