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Sample GSM1099383 Query DataSets for GSM1099383
Status Public on Nov 27, 2013
Title MCL23-1-5 (Puromycin-resistant empty vector), technical rep1
Sample type RNA
 
Source name Control MEFs transfected with puromycin-resistant empty vector (control for MEK1 construct)
Organism Mus musculus
Characteristics treatment: transfected with puromycin-resistant empty vector (control for MEK1 construct)
cell type: Mouse Embryonic Fibroblasts (MEFs)
genotype: [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert]
age: Inmortalized MEFs cultures
cell line: MCL23
Treatment protocol For tamoxifen induction, cultures were treated as appropriate with 4-Hydroxy-tamoxifen (4-OHT, H7904, Sigma-Aldrich) for 6 or 12 days at final concentration 0.6μM to promote the K-Ras locus disruption. Subconfluent cultures of untreated or 4-OHT-treated cell lines were used for RNA extraction.
Growth protocol Cultures of different cell lines were grown in a humidified CO2 (5%) atmosphere at 37°C, in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with fetal bovine serum (10% FBS; Hyclone, Logan, Utah, USA), glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 mg/ml). Hygromycin (200μg/ml, Sigma-Aldrich) or puromycin (2μg/ml, Sigma-Aldrich) was also added as appropriate to MEF cultures expressing BRAF or MEK1, respectively.
Extracted molecule total RNA
Extraction protocol For mRNA expression analysis, total RNA was isolated using the TRIzol reagent and protocol as described by the manufacturer (Ambion, Life Technologies). RNA samples were purified using the RNeasy® Mini Kit (Qiagen) and their concentration, purity and integrity was measured in an “Agilent 2100 Bioanalyzer” (Agilent Technologies). For microRNA studies, total RNA was extracted from two 10cm culture dishes per individual sample by using mirVana miRNA isolation kit (Ambion) according to the manufacturer’s protocol. RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies).
Label Biotin
Label protocol Total RNA was reverse transcribed into double-stranded complementary DNA (cDNA) using an oligo(dT)24 primer containing a T7 polymerase promoterbinding site (Genset). cDNA was then used as a template to synthesize cRNA by in vitro transcription (Ambion T7 Megascript), with incorporation of biotinylated nucleotides (Enzo Diagnostics). For miRNA samples, 1000 ng of total RNA were labeled using the Flash Tag Biotin HSR Labeling kit (Genisphere, P/N HSR10FTA) according to the manufacturer´s instructions.
 
Hybridization protocol For mRNA expression analysis, labeled cRNAs were fragmented and hybridized to the GeneChip Mouse Genome 430 2.0 (Affymetrix) using the Genechip Fluidics Station 450 (Affymetrix). Hybridized arrays were stained with streptavidin–phycoerythrin, rewashed, treated with biotinylated antistreptavidin–phycoerythrin antibodies and restained with streptavidin–phycoerythrin, according to the manufacturer’s protocols. For miRNA samples, hybridizations were performed using the GeneChip miRNA Array (Affymetrix) according to protocols from Affymetrix.
Scan protocol The stained mRNA expression microarrays were finally scanned in a GeneArray Scanner (Hewlett Packard). For miRNA hybridizations, washing and scanning were performed using the Affymetrix GeneChip System (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G).
Description 26. Puromycin-resistant control_miR
miRNA Expression data from K-Raslox cells transfected with puromycin-resistant empty vector
Data processing The RMA function (Irizarry, Hobbs et al. 2003) provided by the package Affy from R/Bioconductor was applied to both mRNA and miRNA raw expression data. The probes were mapped to GENE LOCi by the CDF built by GATExplorer, which can be found at http://bioinfow.dep.usal.es/xgate/mapping/mapping.php
 
Submission date Mar 15, 2013
Last update date Nov 27, 2013
Contact name Alicia Ginel Picardo
E-mail(s) algi@usal.es
Phone 923294801
Fax 923294743
Organization name Cic, Salamanca
Lab Laboratory 1
Street address Campus Miguel de Unamuno
City Salamanca
State/province Select a State or Province
ZIP/Postal code 37007
Country Spain
 
Platform ID GPL8786
Series (1)
GSE45222 Reversible mRNA and miRNA expression patterns in the transcriptome of Rasless fibroblasts

Data table header descriptions
ID_REF
VALUE RMA signal intensity (mice miRNAs probesets)

Data table
ID_REF VALUE
mmu-let-7a-star_st 0.282356195
mmu-let-7a_st 8.626063542
mmu-let-7b-star_st 0.504739192
mmu-let-7b_st 11.08952989
mmu-let-7c-1-star_st 0.749796686
mmu-let-7c-2-star_st 2.108887703
mmu-let-7c_st 10.96671336
mmu-let-7d-star_st 0.083149178
mmu-let-7d_st 10.38969029
mmu-let-7e_st 10.47592806
mmu-let-7f-star_st 1.737555427
mmu-let-7f_st 4.376799685
mmu-let-7g-star_st 0.865195276
mmu-let-7g_st 2.138211617
mmu-let-7i-star_st 0.187502429
mmu-let-7i_st 7.386162115
mmu-miR-1-2-as_st 0.233909019
mmu-miR-100_st 4.877829033
mmu-miR-101a-star_st 0.226558341
mmu-miR-101a_st 0.120325069

Total number of rows: 609

Table truncated, full table size 17 Kbytes.




Supplementary file Size Download File type/resource
GSM1099383_26.G7_miRNA-1_0_.CEL.gz 138.9 Kb (ftp)(http) CEL
Processed data included within Sample table

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