|
| Status |
Public on Jan 31, 2008 |
| Title |
Peritoneal Carcinomatosis of gastric cancer 1- versus 2+ |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Patient No 1 castric cancer without PC
|
| Organism |
Homo sapiens |
| Characteristics |
castric cancer without PC
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Machery & Nagel RNA extraction kit performed according to the provided manual
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl MessageAmp II aRNA Amplification Kit performed according to the provided manual
|
| |
|
| Channel 2 |
| Source name |
Patient No 2 gastric cancer with PC
|
| Organism |
Homo sapiens |
| Characteristics |
gastric cancer with PC
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Machery & Nagel RNA extraction kit performed according to the provided manual
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl MessageAmp II aRNA Amplification Kit performed according to the provided manual
|
| |
|
| |
| Hybridization protocol |
Pre-hybridization
Note: Do not allow the slides to dry during the pre-hybridization and washing
procedure.
1. Pre-warm an appropriate volume of Nexterion 70mer Oligo Pre-Hyb to 48°C for
10 minutes until the precipitate is completely dissolved.
2. Incubate slides in Nexterion 70mer Oligo Pre-Hyb for 45 min at 48°C. Use a large
enough volume of buffer to ensure that all slides are completely covered with
Nexterion Pre-Hyb.
3. During the pre-hybridization step, prepare Wash Solution 1 by diluting Nexterion
70mer Oligo Wash B 1:40 with diH2O. Prepare 50 ml per slide (i.e. 1.25 ml Nexterion
70mer Oligo Wash B, add diH2O to 50 ml).
4. Wash 5 min in Wash Solution 1 at 20 to 25°C.
5. Rinse with diH2O for 30 sec.
6. Dry the Nexterion Slide E with nitrogen.
Hybridization
Prewarm Hybsolution 15 minutes before hybridization at 68°C. Clean coverslips with dH20 and 100% Ethanol and dry the coverslips with nitrogen.
1. Pipette 40µl of hybridization to the solution. Denature the fragmented target in the hyb-buffer by heating at 95°C for 3 min. Then let the probe cool down for 1 min at RT. Afterwards place the target onto the array surface of a prehybridized slide under the coverslip
3. Hybridize the slide at 48°C for 16 hours.
Post-hybridization washing
Note: Do not allow the slides to dry during the hybridization and washing
procedure. Wash Solution 2, 3 and 4 should be prepared in advance. Use at least
50 ml solution per slide.
Post-hybridization washing solutions:
Preparation:
Wash Solution 2: 50 ml Nexterion 70mer Oligo Wash A,
25 ml Nexterion 70mer Oligo Wash B,
Make up to 500 ml with diH2O
Wash Solution 3: 50 ml Nexterion 70mer Oligo Wash A,
Make up to 500 ml with diH2O
Wash Solution 4: 5 ml Nexterion 70mer Oligo Wash A,
Make up to 500 ml with diH2O
1. Place the array into a slide rack and immerse in a dish containing Wash Solution 2.
Wash in the above solution 1 x 10 min at 20 to 25°C.
2. Wash 1 x 10 min in Wash Solution 3 at 20 to 25°C.
3. Wash 1 x 10 min in Wash Solution 4 at 20 to 25°C.
4. Dry the array with nitrogen.
5. Protect the array from light, dust and abrasion of the array surface, until ready for
scanning.
|
| Scan protocol |
not further discription needed
|
| Description |
Slides were scanned in a microarray scanner (Genetix Limited, Hampshire, UK). Photomultiplier tube voltage was set to 100% for both red and green channels. The two resulting green and red images were overlaid using ImaGene 5 (BioDiscovery, Inc., CA, USA).
|
| Data processing |
The data was normalized using loess normalization on the normexp-background corrected expression values, followed by a dye-swap normalization and in-between-array quantile normalization. Both the loess and quantile normalization methods were used as provided in the limma package
|
| |
|
| Submission date |
Aug 02, 2007 |
| Last update date |
Jan 31, 2008 |
| Contact name |
Kay Nieselt |
| E-mail(s) |
kay.nieselt@uni-tuebingen.de
|
| Phone |
(+49) 70712978981
|
| Organization name |
Institute for Bioinformatics and Medical Informatics
|
| Department |
Department for Computer Science
|
| Lab |
Integrative Transcriptomics
|
| Street address |
Sand 14
|
| City |
Tübingen |
| State/province |
DE |
| ZIP/Postal code |
72076 |
| Country |
Germany |
| |
|
| Platform ID |
GPL5676 |
| Series (1) |
| GSE8657 |
Peritoneal Carcinomatosis of gastric cancer |
|
