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Status |
Public on Jan 01, 2013 |
Title |
No.2 vs No.7 Tamoxifen-treated wild type prostaspheres versus Tamoxifen-treated Sox9 prostaspheres |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
No. 2 SOX9
|
Organism |
Mus musculus |
Characteristics |
tissue: prospheres from UGE treatment: Tamoxifen genetic background: C57/Bl6J genotype: SOX9 wild type
|
Treatment protocol |
After 6 days in matrigel plus 10–8 millimolar Dihydrotestosterone, prostaspheres were exposed to Tamoxifen (5 micromolar) or vehicle for 6 additional days. Prostaspheres were then snap frozen in liquid nitrogen.
|
Growth protocol |
C57/Bl6J, ERCre+/+Sox9flox/flox and ERCre+/+Sox9flox/+ urogenital sinus (UGS) were harvested at 16 days post conception. UGS were collected, digested, washed. Epithelial cells then were isolated, minced and incubated in collagenase (1mg/ml) and DNase (1mg/ml). Cells were washed, cultured overnight and isolated into single cells. Prostate epithelial cells were washed, cultured overnight, isolated into single cells, and passed in Matrigel to form prostaspheres.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was prepared from homogenized frozen tissue using the RNeasy system (Qiagen). RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies)
|
Label |
Cy5
|
Label protocol |
250 ng of total RNA input was reverse transcribed into by oligo dT primer incorporating a T7 promoter sequence, and labelled cRNA was obtained by T7 RNA polymerase transcription (Low RNA Input Fluorescent Linear Amplification Kit, Agilent Technologies). cRNA was purified using RNeasy mini kit (Qiagen).
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Channel 2 |
Source name |
No.7 Ctrl
|
Organism |
Mus musculus |
Characteristics |
tissue: prospheres from UGE treatment: Tamoxifen genetic background: C57/Bl6J genotype: Sox9 null
|
Treatment protocol |
After 6 days in matrigel plus 10–8 millimolar Dihydrotestosterone, prostaspheres were exposed to Tamoxifen (5 micromolar) or vehicle for 6 additional days. Prostaspheres were then snap frozen in liquid nitrogen.
|
Growth protocol |
C57/Bl6J, ERCre+/+Sox9flox/flox and ERCre+/+Sox9flox/+ urogenital sinus (UGS) were harvested at 16 days post conception. UGS were collected, digested, washed. Epithelial cells then were isolated, minced and incubated in collagenase (1mg/ml) and DNase (1mg/ml). Cells were washed, cultured overnight and isolated into single cells. Prostate epithelial cells were washed, cultured overnight, isolated into single cells, and passed in Matrigel to form prostaspheres.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was prepared from homogenized frozen tissue using the RNeasy system (Qiagen). RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies)
|
Label |
cy3
|
Label protocol |
250 ng of total RNA input was reverse transcribed into by oligo dT primer incorporating a T7 promoter sequence, and labelled cRNA was obtained by T7 RNA polymerase transcription (Low RNA Input Fluorescent Linear Amplification Kit, Agilent Technologies). cRNA was purified using RNeasy mini kit (Qiagen).
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|
Hybridization protocol |
Fragmentation was carried out by the Sidney Kimmel Comprehensive Cancer Center Microarray Core Facility by incubating at 60 degrees Celsius for 30 minutes and stopped by adding equal volume of 2x hybridization buffer (Agilent Technologies). Fragmented targets were added on Agilent Whole Mouse Genome G4122A Microarrays (Agilent Technologies). The microarrays were assembled into a hybridization chamber (Agilent Technologies) and hybridized at 60 degrees Celsius for 17 hours in a hybridization oven with rotation. Hybridized microarrays were washed and dried according to the Agilent microarray processing protocol.
|
Scan protocol |
Microarrays were scanned by the Sidney Kimmel Comprehensive Cancer Center Microarray Core Facility with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies for expression microarrays with 100% PMT and 10 micometer resolutions. Data was extracted using Feature Extraction Software version 10.5.1.1 (Agilent Technologies).
|
Data processing |
Within-array dye effects were corrected by the 'loess' normalization method. No background subtraction was performed prior to normalization. Positive and negative microarray control features were not used to compute the 'loess' smoothing and were further excluded from subsequent analyses. Data pre-processing was performed using the R/Bioconductor package 'limma'.
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Submission date |
Jan 30, 2012 |
Last update date |
Jan 01, 2013 |
Contact name |
Luigi Marchionni |
E-mail(s) |
marchion@jhu.edu
|
Phone |
410-502-8179
|
Organization name |
Johns Hopkins University
|
Department |
Oncology
|
Lab |
Cancer Biology Program
|
Street address |
1550 Orleans St., CRB2, Room 1M52
|
City |
Baltimore |
State/province |
MD |
ZIP/Postal code |
21231 |
Country |
USA |
|
|
Platform ID |
GPL4134 |
Series (1) |
GSE35419 |
Sox9 mediated differential gene expression was assessed in prostaspheres derived from urogenital sinus epithelial cells |
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