|
| Status |
Public on Dec 08, 2011 |
| Title |
Pooled (Cy3) v PC3M (wt) (Cy5) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled knockdowns
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC3M
|
| Treatment protocol |
Short hairpin RNA for RPL19 cloned into a pSilencer™ 4.1-CMV neo (Ambion) expression vector and transfected into PC3-M prostate cells with SiPORT XP-1 reagent (Ambion)
|
| Growth protocol |
PC3-M prostate cells were grown in RPMI 1640 media containing L-Glutamine (2mM), penicillin (1000units/ml), streptomycin (100μg/ml) and 10% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with a Rneasy mini kit (Qiagen) according to manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Total RNA was primed with 1.2 μL of T7 Promoter Primer at 65°C for 10 min, reversed transcribed at 40°C for 2 h in the presence of 1mM dNTP mix and then labelled with Cyanine 3-CTP or Cyanine 5-CTP according to Agilent instructions
|
| |
|
| Channel 2 |
| Source name |
PC3M
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC3M
|
| Treatment protocol |
Short hairpin RNA for RPL19 cloned into a pSilencer™ 4.1-CMV neo (Ambion) expression vector and transfected into PC3-M prostate cells with SiPORT XP-1 reagent (Ambion)
|
| Growth protocol |
PC3-M prostate cells were grown in RPMI 1640 media containing L-Glutamine (2mM), penicillin (1000units/ml), streptomycin (100μg/ml) and 10% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with a Rneasy mini kit (Qiagen) according to manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Total RNA was primed with 1.2 μL of T7 Promoter Primer at 65°C for 10 min, reversed transcribed at 40°C for 2 h in the presence of 1mM dNTP mix and then labelled with Cyanine 3-CTP or Cyanine 5-CTP according to Agilent instructions
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added and samples were applied to microarrays enclosed in Agilent microarray hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software.
|
| Data processing |
Spatial representations of the hybridization signals were examined to confirm absence of technical artifacts. The distribution of background and foreground signals and pre-normalization MA plots were examined to measure the quality of the hybridization. Low quality spots identified by the Agilent image processing software were not used in the subsequent analyses. Expression signal estimates were derived from the red (cy3) and green (cy5) Agilent Processed Signal data by normalizing using the LOESS algorithm and background correction using a fitted convolution of normal and exponential distributions.
|
| |
|
| Submission date |
Jan 20, 2011 |
| Last update date |
Dec 09, 2011 |
| Contact name |
Ian Giddings |
| E-mail(s) |
ian.giddings@icr.ac.uk, daniel.brewer@icr.ac.uk
|
| Phone |
+44 20 8722 4293
|
| Fax |
+44 20 8722 4141
|
| URL |
http://www.crukdmf.icr.ac.uk
|
| Organization name |
Institute of Cancer Research
|
| Department |
Section of Molecular Carcinogenesis
|
| Lab |
CANCER RESEARCH UK DNA Microarray Facility
|
| Street address |
15 Cotswold Road
|
| City |
Sutton |
| State/province |
Surrey |
| ZIP/Postal code |
SM2 5NG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE26774 |
siRNA knockdown of ribosomal protein gene RPL19 abrogates the aggressive phenotype of human prostate cancer |
|
