|
| Status |
Public on Aug 01, 2006 |
| Title |
P208 HCT-116, DNMT3B knockout, non-treated |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cell line HCT-116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line HCT-116, Ki-RAS-mutation, DNMT3B knockout, non-treated
|
| Biomaterial provider |
Institute of Pathology, University Hospital Charite, Berlin, Germany; cell lines were generated as described previously (Rhee et al., Nature, 2002)
|
| Treatment protocol |
non-treated
|
| Growth protocol |
cells were cultivated in McCoy´s 5A with addition of 10% FCS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted using Qiagen RNeasy Midi Kit reagents; 2µg total RNA was amplified one round based on T7 RNA Polymerase (Ambion Message Amplification Kit Reagents, 6h IVT)
|
| Label |
Cy3
|
| 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), one round amplified
|
| Organism |
Homo sapiens |
| Characteristics |
common reference sample
|
| Biomaterial provider |
Stratagene
|
| Treatment protocol |
none
|
| Growth protocol |
none
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2µg total RNA was amplified one round based on T7 RNA Polymerase (Ambion Message Amplification Kit Reagents, 6h IVT)
|
| 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 Cy5-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 |
P208 HCT-116, DNMT3B knockout, non-treated
|
| Data processing |
raw data processing was performed using ArrayMagic (Buness et al., 2005) and VSN normalization method; two independent array hybridization from every sample were merged; after removal of bad quality clones (marked as - and 0 on array, generalized log2 ratios from 26618 cDNA clones were given in the data table
|
| |
|
| Submission date |
Nov 29, 2005 |
| Last update date |
Jun 05, 2006 |
| Contact name |
Ruprecht Kuner |
| Organization name |
German Cancer Research Center and National Center of Tumor Diseases
|
| Department |
Molecular Genetics
|
| Lab |
Unit Cancer Genome Research
|
| Street address |
Im Neuenheimer Feld 460
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL3050 |
| Series (1) |
| GSE3699 |
DNA-Methyltransferases (DNMT) knockouts |
|
