|
| Status |
Public on Jun 25, 2008 |
| Title |
HPV 16 immortalized keratinocytes (VUMC_MCF26cl4_b2s63) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
foreskin
|
| Organism |
Homo sapiens |
| Characteristics |
HPV 16 immortalized keratinocytes
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol according to manufacturers' instructions
|
| Label |
Cy3
|
| Label protocol |
cDNA was prepared from 75 micrograms of total RNA using oligo-dT20-VN primer (Invitrogen) and coupled to Cy3 by enzymatic labelling with dUTP-Cy3
|
| |
|
| Channel 2 |
| Source name |
universal human reference RNA (Stratagene)
|
| Organism |
Homo sapiens |
| Characteristics |
Pooled RNA of 10 cell lines giving broad gene coverage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
see the Stratagene web site
|
| Label |
Cy5
|
| Label protocol |
as ch1, Cy5 in stead of Cy3
|
| |
|
| |
| Hybridization protocol |
Slides were pre-hybridised for 45 minutes at 37°C with a pre-hybridisation solution containing 30 micrograms of salmon sperm DNA (Gibco), 12 micrograms of poly A (Pharmacia), 60 micrograms of yeast tRNA (Sigma) and 24 micrograms of Cot-1 DNA (Invitrogen) dissolved in 127 microliter hybridisation mix (0.2% SDS, 8% glycerol, 50% formamide and 0.1% dextrane sulphate in 2x SSC). Pre-hybridisation was followed by probe hybridisation for 14 hours at 37°C.
|
| Scan protocol |
as described by Buermans et al, Physiol Genomics, volume 21 (3), p 314-23
|
| Description |
HPV 16 immortalized keratinocytes with ectopic hTERT and chromosome 6, endogenous hTERT negative
|
| Data processing |
Spots were quantified in ImaGene 5.6.1 software using default settings. Microarray data were normalised using Lowess regression. When both intensities were below 50, hybridisation was assumed inefficient and the ratio value was considered ‘missing’. The cut-off “50” is based on technical reproducibility: intensities above 50 were reasonably reproducible for technical replicates on our platform. To obtain more stable ratios, intensity values below 50 were substituted by 50 when the other channel was above 50.
|
| |
|
| Submission date |
Jun 26, 2007 |
| Last update date |
Jun 26, 2007 |
| Contact name |
Daoud Sie |
| E-mail(s) |
d.sie@vumc.nl
|
| Phone |
+31 20 4442428
|
| Organization name |
Vrije Universiteit Medical Center
|
| Department |
Pathology
|
| Lab |
Microarray Core Facility
|
| Street address |
De Boelelaan 1117
|
| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL2902 |
| Series (1) |
| GSE8288 |
Identification of markers associated with deregulated hTERT during HPV-medaited transformation |
|
| Data table header descriptions |
| ID_REF |
|
| Signal Median CH1 |
Spot signal in channel 1 (Cy3) |
| Background Median CH1 |
Background signal channel 1 (Cy3) |
| Signal Median CH2 |
Spot signal in channel 2 (Cy5) |
| Background Median CH2 |
Background signal channel 2 (Cy5) |
| Flag |
A flag to highlight spots that suffer from poor printing and other experimental artefacts (0 acceptable; 1-4 not acceptable) |
| VALUE |
Normalized value of the log2ratio ch1/ch2. Flagged features are excluded. Values where also deleted if the each triplicate had a Standard Deviation higher than 0.2. |
| UNF_VALUE |
Normalized value of the log2ratio. Flagged features are excluded. Values where also deleted if the each triplicate had a Standard Deviation higher than 0.2. |
