|
| Status |
Public on Aug 03, 2012 |
| Title |
EGI1_in_vivo.N |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
EGI-1 xenograft treated with Saracatinib
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EGI-1
cell type: Biliary tract carcinoma (BTC) xenograft
treated with: 25 mg/Kg/die Saracatinib
|
| Treatment protocol |
For in vitro experiment: 24 hours at 10 uM of Saracatinib. In Vivo: 21 days of treatement woth saracatinib at the dose of 25 mg/Kg/die
|
| Growth protocol |
RPMI + 10%FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from EGI-1 cells using TRIZOL (Life Technologies, Gathersburg, MD); in the case of frozen EGI-1 xenograft tissues, RNA was extracted with TriReagent (Sigma, St. Louis, MO, USA) and IKA T25 Digital ULTRA TURRAX homogenizer. For each sample and reference pair, equal amounts of mRNA were amplified by means of the Amino Allyl MessageAmp II aRNA Kit (Ambion Inc., Austin, TX) to obtain amino allyl antisense RNA. One round of amplification was performed and both dsDNA and aaRNA underwent a purification step using columns provided with the kit.
|
| Label |
Cy5
|
| Label protocol |
Labelling was performed using NHS ester Cy3 or Cy5 dyes (GE HealthCare, Buckinghamshire, UK) able to react with the modified RNA.
|
| |
|
| Channel 2 |
| Source name |
EGI-1 xenograft untreated
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EGI-1
cell type: Biliary tract carcinoma (BTC) xenograft
|
| Treatment protocol |
For in vitro experiment: 24 hours at 10 uM of Saracatinib. In Vivo: 21 days of treatement woth saracatinib at the dose of 25 mg/Kg/die
|
| Growth protocol |
RPMI + 10%FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from EGI-1 cells using TRIZOL (Life Technologies, Gathersburg, MD); in the case of frozen EGI-1 xenograft tissues, RNA was extracted with TriReagent (Sigma, St. Louis, MO, USA) and IKA T25 Digital ULTRA TURRAX homogenizer. For each sample and reference pair, equal amounts of mRNA were amplified by means of the Amino Allyl MessageAmp II aRNA Kit (Ambion Inc., Austin, TX) to obtain amino allyl antisense RNA. One round of amplification was performed and both dsDNA and aaRNA underwent a purification step using columns provided with the kit.
|
| Label |
Cy3
|
| Label protocol |
Labelling was performed using NHS ester Cy3 or Cy5 dyes (GE HealthCare, Buckinghamshire, UK) able to react with the modified RNA.
|
| |
|
| |
| Hybridization protocol |
Hybridization with dye-swap duplication was performed to compare sample versus reference. The same quantity of differentially labelled sample and reference was put together, fragmented and hybridized to oligonucleotide glass arrays with sequences representing 60 K human unique genes and transcripts. All steps were performed using the Gene Expression Hybridization kit (Agilent Technologies) following the 60-mer oligo microarray processing protocol (Agilent Technologies).
|
| Scan protocol |
Images were quantified using Agilent Feature Extraction Software (version 9.5).
|
| Description |
Normal
|
| Data processing |
Raw data were processed using the statistical computing software R and packages from Bioconductor (www.bioconductor.org); limma was used for preprocessing and differential expression analysis. Raw intensities were first background corrected (normexp method, with an offset value of 50 added to red and green intensities so that log-ratios are shrunk towards zero at lower intensities). In order to set log-ratios average to zero within each array and to have similar log-ratio distributions across all arrays, loess and Aquantile normalization were performed.
|
| |
|
| Submission date |
Mar 20, 2012 |
| Last update date |
Aug 03, 2012 |
| Contact name |
caterina Peraldo Neia |
| E-mail(s) |
caterina.peraldoneia@gmail.com
|
| Organization name |
Fondazione Edo ed Elvo Tempia
|
| Department |
Laboratory of Cancer Genomics
|
| Lab |
Laboratory of Cancer Genomics
|
| Street address |
via Malta 3
|
| City |
Biella |
| State/province |
Biella |
| ZIP/Postal code |
13900 |
| Country |
Italy |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE36622 |
EGI-1 cell line treated with Saracatinib vs EGI-1 cell line untreated; EGI-1 xenograft treated with Saracatinib vs EGI-1 untreated |
|
