|
| Status |
Public on Jul 01, 2014 |
| Title |
M093243_IU |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Gastrointestinal Tissue with active Crohn's Fistula
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: Female
patient age: 54
clinical comments: Enterocutaneous fistula resected
|
| Treatment protocol |
Samples were selected from patients presenting with Crohn’s Disease fistulae at Eastern Health Department of Gastroenterology and Hepatology (Arnold Street, Box Hill, Victoria, Australia). The 8 samples used in this study were formalin-fixed paraffin-embedded (FFPE) specimens resected as part of a surgical intervention to remove fistula tissue. To focus on changes within the individual patient we used tissue taken from the same surgery at an uninvolved region of the intestine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total gDNA was extracted and RNase treated with the QIAmp® DNA FFPE kit (Qiagen, Australia) following the manufacturer’s instructions. For each sample approximately 8 FFPE sections (20mm x 20mm x 10µm per section) were used. gDNA was further treated using the MiniElute PCR purification kit (Qiagen, Australia) followed by whole genome amplification (WGA) using the Ovation® WGA FFPE System (Nugen, USA). For each WGA 200 ng of starting material was used. The amplified DNA product was purified using the MiniElute PCR purification kit (Qiagen, Australia). Patient samples were interrogated for CNV using 4 x 180K (60-mer) genome-wide microarrays (Agilent Technologies, USA). Sample Quality Analysis, hybridisation and data generation were performed by the Australian Genome Research Facility (WEHI, Melbourne, Australia).
|
| Label |
Cy3
|
| Label protocol |
Human gDNA was quality ascertained using the Nanodrop ND-1000 spectrophotometer and standard agarose gel electrophoresis. A total of 500ng was fragmented and labelled using the ULS cydye (Cy3 or Cy5) coupling method (Agilent – 5190-0419). The cydye incorporation was determined using the Nanodrop ND-1000 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Uninvolved Control Gastrointestinal tissue from same individual as test sample
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: Female
patient age: 54
tissue: uninvolved control tissue for enterocutaneous fistula resected
|
| Treatment protocol |
Samples were selected from patients presenting with Crohn’s Disease fistulae at Eastern Health Department of Gastroenterology and Hepatology (Arnold Street, Box Hill, Victoria, Australia). The 8 samples used in this study were formalin-fixed paraffin-embedded (FFPE) specimens resected as part of a surgical intervention to remove fistula tissue. To focus on changes within the individual patient we used tissue taken from the same surgery at an uninvolved region of the intestine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total gDNA was extracted and RNase treated with the QIAmp® DNA FFPE kit (Qiagen, Australia) following the manufacturer’s instructions. For each sample approximately 8 FFPE sections (20mm x 20mm x 10µm per section) were used. gDNA was further treated using the MiniElute PCR purification kit (Qiagen, Australia) followed by whole genome amplification (WGA) using the Ovation® WGA FFPE System (Nugen, USA). For each WGA 200 ng of starting material was used. The amplified DNA product was purified using the MiniElute PCR purification kit (Qiagen, Australia). Patient samples were interrogated for CNV using 4 x 180K (60-mer) genome-wide microarrays (Agilent Technologies, USA). Sample Quality Analysis, hybridisation and data generation were performed by the Australian Genome Research Facility (WEHI, Melbourne, Australia).
|
| Label |
Cy5
|
| Label protocol |
Human gDNA was quality ascertained using the Nanodrop ND-1000 spectrophotometer and standard agarose gel electrophoresis. A total of 500ng was fragmented and labelled using the ULS cydye (Cy3 or Cy5) coupling method (Agilent – 5190-0419). The cydye incorporation was determined using the Nanodrop ND-1000 spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
The fragmented labelled DNA was then prepared for hybridisation to the Agilent 4x180k Human CGH arrays using the aCGH hybridisation kit (Agilent – 5188-5220) where each hybridisation reaction had a final volume of 110ul. The Agilent hybridisation chambers were prepared according to manufacturer’s instructions. Each sample was loaded on to the Agilent hybridisation gaskets slide which is placed into a hybridisation chamber. The 4x180k array slide was carefully lowered onto the gasket to create a sealed array for each sample. The hybridisation chambers were then placed in a rotating hybridisation oven at 65C for 24 hours.
|
| Scan protocol |
Scanned on an Agilent G2505C scanner.
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
US83403541_252206023757_S01_CGH_107_Sep09_2_1_2
uninvolved control tissue for enterocutaneous fistula resected
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Feb 12, 2013 |
| Last update date |
Jul 01, 2014 |
| Contact name |
phillip david parker |
| Organization name |
Deakin University
|
| Department |
Health
|
| Lab |
CPAN
|
| Street address |
221 Burwood Highway
|
| City |
Burwood |
| State/province |
Victoria |
| ZIP/Postal code |
3125 |
| Country |
Australia |
| |
|
| Platform ID |
GPL10123 |
| Series (1) |
| GSE44275 |
Analysis of whole-genome copy-number variation in Crohn's disease fistula tissue |
|
