|
| Status |
Public on Apr 22, 2008 |
| Title |
Dicer1-null 27G5 cells overexpressing Dnmt3a,3b versus Dicer1-null 27G5 cells 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
27 g5+DNMT3a,3b
|
| Organism |
Mus musculus |
| Characteristics |
Dicer1-null 27G5 cells overexpressing Dnmt3a and 3b
|
| Treatment protocol |
No special treatment were applied, cells were growth in appropriate conditions (see growth protocol) and harvested to prepare RNA.
|
| Growth protocol |
Cell lines wild type and Dicer-1 (cultures 27G5 and 27H10) or Dicer-1 null (27G5 or 27H10) + DNMT1 and Dicer-1 null (27G5 and 27H10) + DNMT3a3b, were cultivated on gelatin-coated plates with media supplemented with Leukemia Inhibitory Factor.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Commercial RNeasy kit (Qiagen) was used in spin-column format by following manufacturer instructions. Total RNA was analysed by Lab-chip technology on an Agilent 2100 Bioanalyzer. Samples' RNA Integrity Numbers were in the range 8.0 to 9.5
|
| Label |
Cy3
|
| Label protocol |
Amount of nucleic acid labeled: 2ug.Amplification: by RNA polymerases.Commercial "Two-Color Microarray-Based Gene Expression Analysis" kit by following manufacturer instructions. Agilent manual G4140-90050 Ver. 5.5 Feb 2007. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Dicer Null 27G5
|
| Organism |
Mus musculus |
| Characteristics |
Dicer-1 null 27G5 cells
|
| Treatment protocol |
No special treatment were applied, cells were growth in appropriate conditions (see growth protocol) and harvested to prepare RNA.
|
| Growth protocol |
Cell lines wild type and Dicer-1 (cultures 27G5 and 27H10) or Dicer-1 null (27G5 or 27H10) + DNMT1 and Dicer-1 null (27G5 and 27H10) + DNMT3a3b, were cultivated on gelatin-coated plates with media supplemented with Leukemia Inhibitory Factor.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Commercial RNeasy kit (Qiagen) was used in spin-column format by following manufacturer instructions. Total RNA was analysed by Lab-chip technology on an Agilent 2100 Bioanalyzer. Samples' RNA Integrity Numbers were in the range 8.0 to 9.5
|
| Label |
Cy5
|
| Label protocol |
Amount of nucleic acid labeled: 2ug.Amplification: by RNA polymerases.Commercial "Two-Color Microarray-Based Gene Expression Analysis" kit by following manufacturer instructions. Agilent manual G4140-90050 Ver. 5.5 Feb 2007. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Chamber type: SureHyb hybridization chamber (Agilent). Quantity of labeled extract used: 825 ng. Duration: 17 hours.Volume: 100 uL.Temperature (ºC): 65.
|
| Scan protocol |
Scanned on an G2565BA DNA microarray scanner (Agilent). Images were quantified using Agilent Feature Extraction Software (ver. 9.5.1).
|
| Description |
no additional information
Dye swap technical replicate of MT3a3b / Null27g5
|
| Data processing |
Per Spot: Divide by control channel. Each gene's measured intensity was divided by its control channel value in each sample; if the control channel was below 0 then 0 was used instead. If the control channel and the signal channel were both below 0 then no data was reported.
Per Chip: Normalize to 50th percentile. Each measurement was divided by the 50,0th percentile of all measurements in that sample.The percentile was calculated using only genes marked present.
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| |
|
| Submission date |
Apr 21, 2008 |
| Last update date |
Apr 22, 2008 |
| Contact name |
Roberta Benetti |
| E-mail(s) |
roberta.benetti@lncib.it
|
| Organization name |
CNIO
|
| Street address |
Calle Melchor Fernandez Almagro
|
| City |
Madrid |
| ZIP/Postal code |
28003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE11229 |
Dicer-null cell lines vs. wt ES cells and Dicer-null cell lines vs. Dicer-null cells overexpressing Dnmt1 or Dnmt3a/3b |
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