|
| Status |
Public on Nov 27, 2013 |
| Title |
DU315-6-12d-4OHT-S.r. (Rasless), technical rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Control cells after 12 days of 4OHT treatment becaming Rasless MEFs
|
| Organism |
Mus musculus |
| Characteristics |
treatment: 12 days of 4OHT treatment becaming Rasless MEFs
cell type: Mouse Embryonic Fibroblasts (MEFs)
genotype: [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert]
age: Inmortalized MEFs cultures
cell line: DU315
|
| 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 |
14. Rasless MEFs
Gene Expression data from Rasless sample: 4-OHT-treated K-Raslox cells during 12 days (non-proliferating cells)
|
| 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
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| |
|
| 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 |
GPL1261 |
| Series (1) |
| GSE45222 |
Reversible mRNA and miRNA expression patterns in the transcriptome of Rasless fibroblasts |
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