|
| Status |
Public on Aug 14, 2017 |
| Title |
MCF7_shRNA_PRH_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7_shRNA_PRH knockdown
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
gender: Female
tissue source: Breast adenocarcinoma
transfected with: PRH shRNA lentiviral construct
|
| Treatment protocol |
Knockdown of PRH in MCF-7 cells was performed using an isopropyl β-D-1-thiogalactopyranoside-inducible PRH shRNA lentiviral construct (TRCN0000274008 Sigma). Cells were infected with the PRH shRNA lentiviral construct or a control scrambled shRNA lentivirus (SHC332) and after 48 h transduced cells were selected using puromycin. Stably transduced cell lines were grown in the presence of 1 mM isopropyl β-D-1-thiogalactopyranoside for 7 days to induce shRNA expression.
Overexpression of PRH was performed by infection of MCF-7 cells with an adenovirus expressing human PRH tagged with the Myc9E10 epitope (moi 50). Control cells were infected with emtpy adenoviral vector (moi 50). RNA was extracted 48 hours after infection.
|
| Growth protocol |
MCF7 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen) and DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). RNA was converted to double-stranded cDNA using cDNA synthesis kit (Roche Nimblegen, 11 117 831 001)
|
| Label |
cy3
|
| Label protocol |
Samples were labelled with Cy3 using a Nimblegen One Colour Labeling Kit (Roche Nimblegen, 06 370 438 001)
|
| |
|
| Hybridization protocol |
Samples were hybridised using a Nimblegen Hybridisation Kit (Roche Nimblegen, 05 583 934 001), including Sample Tracking Controls (Roche Nimblegen, 05 223 512 001)
|
| Scan protocol |
Slides were scanned on a Nimblegen MS200 microarray scanner at a resolution of 2µm with balanced Speed/Sensitivity and 100% laser intensity using autogain
|
| Description |
SAMPLE 5
|
| Data processing |
Data were extracted using DEVA (Roche Nimblegen) and normalised by robust multichip average in R.
|
| |
|
| Submission date |
Feb 14, 2017 |
| Last update date |
Aug 14, 2017 |
| Contact name |
John Halsall |
| E-mail(s) |
j.halsall@bham.ac.uk
|
| Organization name |
University of Birmingham
|
| Department |
Cancer and Genomic Sciences
|
| Lab |
Chromatin & Gene Expression Group
|
| Street address |
Medical School, Vincent Drive
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18943 |
| Series (1) |
| GSE94865 |
Gene expression changes following knockdown or overexpression of HHEX/PRH |
|
