|
| Status |
Public on May 31, 2008 |
| Title |
Mice_c-myc trans, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Lung Adenocarcinomas of c-Myc-Transgenic Female Mice
|
| Organism |
Mus musculus |
| Characteristics |
Genotype: c-Myc transgenic
c-Myc transgenic female mice displayed morphological alterations with varying degree of nuclear atypia, such as bronchiolo-adenomas and bronchiolo-adenocarcinomas. Thus, different stages of malignant transformation of alveolar epithelium were observed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Transcriptome analysis was done according to the manufacturer's recommendation (Affymetrix GeneChip® Expression Analysis Technical Manual (Santa Clara, CA)), using the GeneChip® Test Arrays and GeneChip® Mouse Genome 430 2.0. The frozen lung tissues (10-15 mg) were disrupted and homogenized using a rotor-stator homogenizer, and total RNA was isolated from the tissues using the RNeasy total RNA isolation kit (QIAGEN). RNA of individual samples was pooled as described above, and a second cleanup of isolated RNA was done using the RNeasy Mini Kit (QIAGEN). RNA was checked for quantity, purity, and integrity of the 18S and 28S ribosomal bands by capillary electrophoresis using the NanoDrop ND-1000 and the Agilent 2100 Bioanalyzer.
|
| Label |
biotin
|
| Label protocol |
8 µg of total RNA were used as starting material to prepare cDNA. Synthesis of double-stranded cDNA was done with the GeneChip® one-cycle cDNA Kit (Affymetrix). The cleanup of double-stranded cDNA was done using the GeneChip® Sample Cleanup module (Affymetrix). 12 µl of cDNA solution were used for in vitro transcription. The in vitro transcription was conducted with the GeneChip® IVT Labeling Kit (Affymetrix). The total amount of the reaction product was purified with the GeneChip® Sample Cleanup module (Affymetrix). Purified cRNA was quantified and checked for quality using the NanoDrop ND-1000 and the Agilent 2100 Bioanalyzer. Purified cRNA was cleaved into fragments of 35-200 bases by metal-induced hydrolysis. The degree of fragmentation and the length distribution of the fragmented biotinylated cRNA were checked by capillary electrophoresis using the Agilent 2100 Bioanalyzer.
|
| |
|
| Hybridization protocol |
10 µg of biotinylated fragmented cRNA were hybridized onto the GeneChip® Mouse Genome 430 2.0 array according to the manufacturer's recommendation. The hybridization was performed for 16 hours at 60 rpm and 45 °C in the GeneChip® Hybridization Oven 640 (Affymetrix). Washing and staining of the arrays was done on the GeneChip® Fluidics Station 400 (Affymetrix) according to the manufacturer's recommendation. The antibody signal amplification, washing, and staining protocol (Affymetrix) was used to stain the arrays with streptavidin R-phycoerythrin (SAPE; Invitrogen, USA). To amplify staining, SAPE solution was added twice with a biotinylated anti-streptavidin antibody (Vector Laboratories, CA) staining step in between.
|
| Scan protocol |
The arrays were scanned using the GeneChip® Scanner 3000. Scanned image files were visually inspected for artifacts and then analyzed, each image being scaled to the same target value for comparison between chips. The GeneChip® Operating Software (GCOS) was used to control the fluidics station and the scanner, to capture probe array data, and to analyze hybridization intensity data. Default parameters provided in the Affymetrix data analysis software package were applied for analysis.
|
| Description |
Mice_c-myc trans, biological rep1
|
| Data processing |
For the comparison analysis, it was ensured that the scale factors for the compared chips did not differ by a factor larger than 3. In this study, with four replicates per group, 16 comparison analyses (4 transgenic versus 4 non-transgenic) were conducted. Comparison ranking analysis was additionally done to study concordance between increase calls (I) or decrease calls (D) for replicates (this is counting the number of I-calls and D-calls out of 16). The unpaired one-sided t-test converting the p-value to a two-sided p-value was used to determine the direction and significance of change in a transcript's expression level (Data Mining Tool, version 3.1). Signal values of each group were used as basis for calculation, with the original p-value cutoff determined to be 0.05. To select up-regulated genes in the c-Myc experiments, the following criteria were applied for the comparison conducted: the average signal value of the treatment had to be higher than 100; 4 P-calls had to be in the 4 treatment arrays; the fold change had to be 2.0 or higher (ratio of average signal values); the result of the t-test had to be an Up-change call (p-value < 0.05) (based on single signal values of 4 replicates); there had to be more than 13 (out of 16) induced calls in the comparison ranking.
|
| |
|
| Submission date |
Mar 26, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Jürgen Borlak |
| E-mail(s) |
[email protected]
|
| Phone |
+49-511 5350-520
|
| Fax |
+49-511 5350-573
|
| URL |
http://www.item.fraunhofer.de
|
| Organization name |
Fraunhofer Institut
|
| Street address |
Nikolai-Fuchs-Strasse 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE10954 |
Transcription Profiling of Lung Adenocarcinomas of c-Myc-Transgenic Mice |
|
| Relations |
| Reanalyzed by |
GSE119085 |
