|
| Status |
Public on Jul 01, 2007 |
| Title |
HPV18-positive Hela cell line, siRNA 18E6E7 mediated knockdown_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cell line Hela siRNA 18E6E7_3.5 Rep3
|
| Organism |
Homo sapiens |
| Characteristics |
construct siRNA 18E6E7_3.5, target sequence 5’-CCACAACGUCACACAAUGU-3’ (HPV18 nt 755-773), cell culture replicate 3
|
| Biomaterial provider |
Felix Hoppe-Seyler, Molecular Therapy of Virus-Associated Cancers Group, German Cancer Research Center, Heidelberg, Germany
|
| Treatment protocol |
Synthetic siRNAs (Dharmacon Research, Lafayette, USA) were transfected with Oligofectamine (Invitrogen, Karlsruhe, Germany)
|
| Growth protocol |
cells were cultivated in Dulbecco’s minimal essential medium (D-MEM, pH 7,2, Gibco BRL, Gaithersburg, MD, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987); 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit) according to the manufacturer’s instructions
|
| 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 |
cell line Hela siRNA siControl Rep3
|
| Organism |
Homo sapiens |
| Characteristics |
siRNA siControl, target sequence 5’-UAGCGACUAAACACAUCAA-3’ (Dharmacon Research, Lafayette, USA), cell culture replicate 3
|
| Biomaterial provider |
Felix Hoppe-Seyler, Molecular Therapy of Virus-Associated Cancers Group, German Cancer Research Center, Heidelberg, Germany
|
| Treatment protocol |
Synthetic siRNAs (Dharmacon Research, Lafayette, USA) were transfected with Oligofectamine (Invitrogen, Karlsruhe, Germany)
|
| Growth protocol |
cells were cultivated in Dulbecco’s minimal essential medium (D-MEM, pH 7,2, Gibco BRL, Gaithersburg, MD, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987); 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit) according to the manufacturer’s instructions
|
| 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.
|
| |
|
| |
| 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 |
siRNA18E6E7_DKFZ Replicate 3
|
| Data processing |
raw data processing was performed using ArrayMagic (Buness et al., 2005) including VSN normalization method; after removal of bad quality clones, generalized log ratios from 26629 cDNA clones were given in the data table
|
| |
|
| Submission date |
Jan 30, 2007 |
| Last update date |
Apr 16, 2007 |
| 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) |
| GSE6926 |
Identification of downstream targets of the human papillomavirus E6 and E7 oncoproteins by RNA interference |
|
