|
| Status |
Public on Apr 01, 2013 |
| Title |
colorectal control epithelium_sporadic_biol repl3 |
| Sample type |
RNA |
| |
|
| Source name |
colorectal control epithelium_sporadic
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J-ApcMin/+/J
gender: male
tissue: tumor-free distal colon epithelium
animals per sample: 4
experimental block: 2
|
| Treatment protocol |
For epithelial purification, distal colonic specimens were incubated in prewarmed HBSS supplemented with EDTA (2mM), EGTA (1mM), DTT, and FCS (1%) with gentle agitation at 37°C for 20min. To avoid bias caused by the 20min incuabtion, all tumors were treated exact the same.
|
| Growth protocol |
Colitis-associated tumorigenesis was performed by intraperitoneal injection of Azoxymethane (10mg/kg) into C57BL/6J wildtype mice followed by 3 cycles of Dextran Sodium Sulfate (DSS) in drinking water. Each DSS-cycle was composed of DSS (2.5% (w/v) in drinking water for 7 days, followed by a recovery phase with regular drinking water for 14 days. Sporadic tumors were from C57BL/6J-ApcMin/+/J mice. All tumors were obtained from the from the lower 6th of the large intestine and they had the same size covering between ¼ and up to ½ of the colonic circumferenc as evaluated by mini-endoscopy.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-purification, tumors and purified distal colon epithelium were placed in RNeasy lysis buffer (Qiagen), disrupted and homogenized using a master mill (Qiagen) before being processed by using the RNeasy Mini kit (Qiagen) according to the manufacturer´s recommendations.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared from 5 ug total RNA according to standard protocols. First strand cDNA synthesis was performed with T7-oligo(dT) primer (Invitrogen), reverse transcriptase Superscript II (Invitrogen) ans first strand buffer (Invitrogen). Second strand cDNA synthesis was done with E. coli DNA ligase (Invitrogen), E. coli DNA Polymerase I (Invitrogen) and second strand reaction buffer (Invitrogen). Biotinylated cRNA was synthesized with GeneChip IVT labeling kit (Affymetrix) according to the manufacturer´s instructions. All recommended cleanup-steps were done with GeneChip cleanup module (Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at MOE430A Affymetrix Mouse Expression 430A Array in the GeneChip Hybridization oven 640. Miroarrays were washed and stained according to manufacturer´s recommendations using Streptavidin Phycoerythrin for labeling of hybridized biotinylated cRNA.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner.
|
| Description |
sporCRC contr3
|
| Data processing |
Data processing and analysis was performed with ArrayAssist software 5.0 (Stratagene). GC-RMA method was used for background transformation and normalization of expression levels. For enhanced significance analysis, microarrays were assigned to the 4 groups (CAC, CAC control epithelium, sporadic CRC, sporadic CRC control) according to the origin of the tissue. Baseline transformation (subtraction) was performed in respect to the expression levels of microarrays with control epthelium. Enhanced significance analysis was done with unpaired t-test including Benjamini-Hochberg false discovery rate p-value correction ['enhanced_significance_analysis.txt' available on Series records].
|
| |
|
| Submission date |
Jan 08, 2013 |
| Last update date |
Apr 01, 2013 |
| Contact name |
Clemens Neufert |
| Organization name |
Friedrich-Alexander-University Erlangen-Nürnberg
|
| Department |
First Department of Medicine
|
| Street address |
Ulmenweg 18
|
| City |
Erlangen |
| ZIP/Postal code |
91054 |
| Country |
Germany |
| |
|
| Platform ID |
GPL339 |
| Series (1) |
| GSE43338 |
Gene expression profiling of colitis-associated and sporadic colorectal tumors in mice |
|
