|
| Status |
Public on Aug 15, 2014 |
| Title |
NTera-2 cells SOX2 siRNA, biological replicate 1_miRNA |
| Sample type |
RNA |
| |
|
| Source name |
NTera-2 cells transfected with Ambion Silencer Select SOX2 siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonal carcinoma
cell line: NTera-2
passage: 40-50
|
| Treatment protocol |
24 hours prior to transfection, 2102Ep and NTera-2 cells were seeded on 6-well plates in normal growth medium without antibiotics. For transfection, old media were replaced with reduced-serum Opti-MEM I medium. siRNA previously incubated with Lipofectamine 2000 (2102Ep cells) or Lipofectamine RNAiMAX (NTera-2 cells) in Opti-MEM I were added to the cells and these were incubated under transfection conditions for 6 hours. After transfection, media were replaced with normal growth medium and sample incubated for 72 hours with daily medium refreshing.
|
| Growth protocol |
2102Ep and NTera-2 cell lines were grown in normal growth medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% foetal bovine serium and 2% Penicillin-Streptomycin mixture. Cells were kept at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were extracted with the Ambion miRVana PARIS kit as per manufacturers protocol.
|
| Label |
FAM
|
| Label protocol |
cDNA was prepared from total RNA using the Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit and the Applied Biosystems Megaplex RT Human Pool A and RT Human Pool B primer mix. Further pre-amplification was performed with Applied Biosystems TaqMan PreAmp Master Mix combined with the Applied Biosystems Megaplex PreAmp Primers, Human Pool A and Human Pool B. For PCR, the Applied Biosystems TaqMan Array Human MicroRNA A & B cards were used with Applied Biosystems TaqMan Universal Master Mix, No Amperase® UNG. All protocols were performed as per manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
not applicable
|
| Scan protocol |
qPCR and scanning was performed with an Applied Biosystems Prism 7900HT Sequence Detection System
|
| Data processing |
Ct values were obtained with Applied Biosystems RQ Manager software and were further analysed using Applied Biosystems DataAssist 3.0 software
2^-ddCt method was used to obtain fold changes
|
| |
|
| Submission date |
Jul 14, 2014 |
| Last update date |
Aug 15, 2014 |
| Contact name |
Sebastian Vencken |
| E-mail(s) |
venckens@tcd.ie
|
| Organization name |
University College Dublin
|
| Department |
Clinical Research Centre
|
| Street address |
Catherine McAuley Centre, Nelson Street
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 7 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL18942 |
| Series (2) |
| GSE59380 |
MiRNA expression data from SOX2 knock-out 2102Ep and NTera-2 human embryonal carcinoma cell lines |
| GSE59381 |
Differential gene and microRNA expression in two SOX2-silenced human embryonal carcinoma cell lines |
|
