|
| Status |
Public on Mar 31, 2010 |
| Title |
Patient ID 40 Gleason grade 8 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
patient tissue
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Gleason grade 8
|
| Biomaterial provider |
Prof.Klocker, University of Innsbruck
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolated from laser-capture microdissected epithelial prostate tumor cells of tissues from patients who had undergone radical prostatectomy were subjected to a two-round amplification using the MessageAmpTM II aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, USA).
|
| Label |
Cy5
|
| Label protocol |
direct fluorescent labelling of 2 µg amplified RNA, anneling of 0.5µg Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dATP, 0.4 mM dTTP, 0.24 mM dCTP, 0.125 mM Cy3-dCTP. After hydrolysis of RNA in 0.2 M NaOH, Cy3-labeled probes were purified with Microcon YM-30 column.
|
| |
|
| Channel 2 |
| Source name |
Universal human reference RNA (Stratagene), two rounds amplified
|
| Organism |
Homo sapiens |
| Characteristics |
sample: common reference
|
| Biomaterial provider |
Stratagene
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2 µg total RNA was amplified two-rounds using the MessageAmpTM II aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, USA)
|
| Label |
Cy3
|
| Label protocol |
direct fluorescent labelling of 2 µg amplified RNA, anneling of 500ng Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dTTP, 0.4 mM dATP, 0.24 mM dCTP, 0.125mM Cy-5 dCTP . After hydrolysis of RNA in 0.2 M NaOH, Cy5-labeled probes were purified with Microcon YM-30 column.
|
| |
|
| |
| Hybridization protocol |
ch1 and ch2 together are solved in 50 µl 1x DIG-Easy hybridization buffer (Roche Diagnostics, Mannheim, Germany), containing 10x Denhardt’s solution and 2 ng/µl Cot1-DNA (Invitrogen); sample was denaturated at 65°C for 2min, hybridzation of arrays were performed in Corning chambers 14h at 37°C; slides were washed twice in 1xSSC+0.1SDS and once in 0.1xSSC+ 0.1SDS before air pressure drying and scanning.
|
| Scan protocol |
arrays were scanned with the GenePix 4000B microarray scanner and analyzed using GenePix Pro 4.1 software (Axon Instruments)
|
| Description |
patient.ID 40
|
| Data processing |
raw data processing was performed using ArrayMagic (Buness et al., 2005) and VSN normalization method; Low quality measurements were excluded from further analysis. generalized log2 ratios (test/reference) from the remaining cDNA clones were given in the data table
|
| |
|
| Submission date |
Mar 31, 2009 |
| Last update date |
Apr 02, 2009 |
| Contact name |
Nicole Chui Pressinotti |
| E-mail(s) |
n.pressinotti@dkfz-heidelberg.de
|
| Organization name |
DKFZ
|
| Street address |
Im Neuenheimer Feld 580
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL3050 |
| Series (1) |
|
