|
| Status |
Public on Feb 17, 2007 |
| Title |
thymus_control5 |
| Sample type |
RNA |
| |
|
| Source name |
wild type mouse
|
| Organism |
Mus musculus |
| Characteristics |
this mouse had a mixed gene background
|
| Biomaterial provider |
from Fotini Gounari's lab at Molecular Oncology Research Institute of Tufts-New England Medical Center
|
| Treatment protocol |
thymocytes were kept in TRIZOL reagent and stored at -80C before total RNA isolation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total thymocytes or lymphomas from mice were collected and homogenized for total RNA isolation with TRIZOL reagent (GIBCO BRL) and purified with Qiagen RNeasy mini kit.
|
| Label |
biotin
|
| Label protocol |
The standard protocol for Affymetrix gene chips was used. Briefly, biotinylated cRNA was hybridized onto chips in hybridization buffer overnight at 45C. The chips were then transferred to a fluidics instrument that performs washes to remove cRNA that did not hybridize to its complementary oligonucleotide probe. The bound cRNA was then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors were then added using biotinylated anti-streptavidin antibody and additional SAPE. Each cRNA bound at its complementary oligonucleotide was excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions were captured.
|
| |
|
| Hybridization protocol |
The standard protocol for Affymetrix gene chips was used. Briefly, biotinylated cRNA was hybridized onto chips in hybridization buffer overnight at 45C. The chips were then transferred to a fluidics instrument that performs washes to remove cRNA that did not hybridize to its complementary oligonucleotide probe. The bound cRNA was then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors were then added using biotinylated anti-streptavidin antibody and additional SAPE. Each cRNA bound at its complementary oligonucleotide was excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions were captured.
|
| Scan protocol |
GeneChip HT as scanner
|
| Description |
Total RNA from thymocytes or tumor was extracted using TRIZOL reagent followed by purification with the Qiagen RNeasy mini kit. The quantity and quality of RNA were determined by spectrophotometric analysis and electrophoresis on a 2.0% agarose gel. 10 micrograms of total RNA in 10ul sterile H2O were used for first and second strand cDNA synthesis, followed by an in vitro transcription reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphatases to produce biotinylated cRNAs.
|
| Data processing |
Normalization. Normalization of expression values ensures the comparability of gene expression estimates across different samples. In dChip, normalization is done at Perfect Match (PM) and MM (Mismatch) before the calculation of Model-Based Expression Indices (MBEIs). By default an array with median intensity is chosen as the baseline array against which other arrays are normalized at probe intensity level. The invariant set normalization method is used in which a subset of Perfect Match (PM) probes with small within-subset rank difference in the two arrays is used as a basis for the normalization.
Transformation. We used D-Chip software to calculate expression index. Model based expression index based on PM/MM difference were calculated for each gene in each sample. The details could be found in (C Li and W Wong. Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection. Proc. Natl. Acad. Sci. Vol. 98, 31-36, 2001a.). Using the combination of a pair of 25-mer probes that are designed to be perfect match (PM) and mismatch (MM) to the targets offers the balance of highest sensitivity and specificity in the presence of a complex background-especially for low abundance transcripts.
Data selection procedures. Before analysis, the arrays were normalized at PM/MM probe level followed by computing model-base expression levels. Genes were filtered to adjust variation across samples: 0.30, Standard deviation/Mean, 10.00, P call % > =50%. Genes that showed more than 3-fold change in the thymocytes between LckCre to CD4Cre-Ctnnbex3 or CD4Cre-Ctnnbex3 pretransformed thymocytes to lymphomas were selected. Notch pathway related genes were chosen.
|
| |
|
| Submission date |
Feb 09, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Fotini Gounari |
| E-mail(s) |
fgounari@tufts-nemc.org
|
| Organization name |
Tufts-New England Medical Center
|
| Department |
molecular oncology research institute
|
| Lab |
Fotini Gounari
|
| Street address |
75 Kneeland, 12th floor
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02111 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE7050 |
Stabilization of b-catenin induces lymphomas |
|
| Relations |
| Reanalyzed by |
GSE119085 |
