|
| Status |
Public on Mar 18, 2015 |
| Title |
48h_miR mimic-transfected LNCaP versus control LNCaP 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
48h control LNCaP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
cell type: prostate cancer cells
transfection: 50nM negative control
time: 48h
|
| Treatment protocol |
LNCaP cells were transfected using Lipofectamine2000 (LifeTech) with 50 nM miR mimic or negative control for 24h and 48h.
|
| Growth protocol |
6x10^5 LNCaP cells were plated onto 25cm2 flasks in antibiotic-free RPMI-1640 medium containing 10% fetal calf serum for 72 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using TriPure reagent (ROCHE) following the 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 miR mimic-transfected LNCaP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
cell type: prostate cancer cells
transfection: 50nM hsa-miR-135a mimic
time: 48h
|
| Treatment protocol |
LNCaP cells were transfected using Lipofectamine2000 (LifeTech) with 50 nM miR mimic or negative control for 24h and 48h.
|
| Growth protocol |
6x10^5 LNCaP cells were plated onto 25cm2 flasks in antibiotic-free RPMI-1640 medium containing 10% fetal calf serum for 72 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using TriPure reagent (ROCHE) following the 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 an Agilent Gasket slide coverslip (Agilent hybridization chamber, G2534A, Agilent) in a rotative Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using two color scan setting (for both Cy3 and Cy5 channels) for 4x44k array slides.
|
| Description |
US45103047_252665217420_S01_GE2_107_Sep09_1_4
Dual swap 48h-2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5 (Agilent) using default parameters. Data files (txt format) were generated. Data were normalized using R and limma library. Briefly, background was subtracted using the correction Half with an offset=100. Then Loess normalization and between array normalization with quantile were performed.
|
| |
|
| Submission date |
Mar 28, 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 |
GPL10332 |
| Series (2) |
| GSE45620 |
Identification of hsa-miR-135a target genes in the prostate cancer cell line LNCaP |
| GSE45903 |
Identification of hsa-miR-135a target genes in LNCaP and HeLa cell lines |
|
