|
| Status |
Public on Mar 18, 2015 |
| Title |
48h_miR mimic-transfected HeLa versus control HeLa 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
48h miR mimic-transfected HeLa
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
transfection: hsa-miR-135a mimic
time: 48h
|
| Treatment protocol |
HeLa cells were then transfected using Lipofectamine2000 (LifeTech) with 50 nM miR mimic or negative control for 24h and 48h.
|
| Growth protocol |
3x10^5 HeLa cells were plated onto 6-well plates in antibiotic-free DMEM medium containing 10% fetal calf serum for 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using TriPure reagent (ROCHE) following manufacturer's instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
1 µg of total RNA was amplified with the Amino Allyl MessageAmp II aRNA kit (Ambion) according to the manufacturer's instructions. Fluorescent probes were synthesized by chemical coupling of 5 µg of aminoallyl aRNA with cyanine Cy3 or Cy5 dyes. Fluorescent probes were purified using the RNeasy mini kit (Qiagen). Dye incorporation and aRNA yield were checked with the Nanodrop ND-1000 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
48h control HeLa
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
transfection: 50nM negative control
time: 48h
|
| Treatment protocol |
HeLa cells were then transfected using Lipofectamine2000 (LifeTech) with 50 nM miR mimic or negative control for 24h and 48h.
|
| Growth protocol |
3x10^5 HeLa cells were plated onto 6-well plates in antibiotic-free DMEM medium containing 10% fetal calf serum for 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using TriPure reagent (ROCHE) following manufacturer's instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
1 µg of total RNA was amplified with the Amino Allyl MessageAmp II aRNA kit (Ambion) according to the manufacturer's instructions. Fluorescent probes were synthesized by chemical coupling of 5 µg of aminoallyl aRNA with cyanine Cy3 or Cy5 dyes. Fluorescent probes were purified using the RNeasy mini kit (Qiagen). Dye incorporation and aRNA yield were checked with the Nanodrop ND-1000 spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
1 µg of Cy3/Cy5 dye-labelled aRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55µL containing 2x Agilent fragmentation buffer and 10X Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55µL of 2x Agilent hybridization buffer was added to the final mixture. Combinated Cy3/Cy5 dye-labelled aRNA were hybridized competitively to Agilent Whole Human Genome 4x44k V2 oligonucleotides microarray slides for 18 h at 62 °C in hybridization buffer (5185-5973, Agilent) and under Agilent Gasket slide coverslip (Agilent hybridization chamber, G2534A, Agilent) in a rotative Agilent hybridization oven.
|
| Scan protocol |
Fluorescent microarray TIFF images were collected for both Cy3 and Cy5 by scanning washed dried glass slides with a GenePix 4000B microarray scanner (Axon Instruments).
Image intensity data were extracted and analysed with GenePix Pro6.0 microarray analysis software. After visual inspection, spots of insufficient quality were flagged and GenePix data files (gpr format) were finally generated.
|
| Description |
25148504041_48hmiRCy3
Dual swap 48h-1.
|
| Data processing |
Signal intensities were log2-transformed and normalization was performed by the intensity-dependent Lowess method. Data were imported and analyzed into the R Bioconductor software in order to identify genes with significant change in expression in response to miR mimic transfection.
|
| |
|
| Submission date |
Apr 09, 2013 |
| Last update date |
Mar 18, 2015 |
| Contact name |
Nathalie ALLIOLI |
| E-mail(s) |
Nathalie.Allioli@recherche.univ-lyon1.fr
|
| Phone |
33(0)426235934
|
| Fax |
33(0)426235900
|
| Organization name |
Ecole Normale Supérieure de Lyon (ENS)
|
| Department |
Institut de Génomique Fonctionnelle de Lyon (IGFL)
|
| Lab |
équipe Oncogenèse et Développement
|
| Street address |
46 Allée d'Italie
|
| City |
Lyon Cedex 07 |
| ZIP/Postal code |
69364 |
| Country |
France |
| |
|
| Platform ID |
GPL7038 |
| Series (2) |
| GSE45902 |
Identification of hsa-miR-135a target genes in the HeLa cell line |
| GSE45903 |
Identification of hsa-miR-135a target genes in LNCaP and HeLa cell lines |
|
