|
| Status |
Public on Jun 01, 2007 |
| Title |
P1_P_HSPC |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
P1_P_HSPC
|
| Organism |
Homo sapiens |
| Characteristics |
Hormone-sensitive (or-naïve) prostate cancer cells that were microdissected from the radical prostatectomy sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100,00-200,000 tumor cells or normal cells were isolated selectively using the EZ cut system with a pulsed ultraviolet narrow beam-focus laser (SL Microtest GmbH, Germany) in accordance with the manufacturer’s protocols. Total RNAs were isolated from captured cells with QIAGNE RNAeasy Kit (QIAGEN). After DNAse I treatment, total RNAs were subjected to two rounds of T7-based amplification using Ampliscribe T7 Transcription Kit (Epicentre Technologies), or MEGAscript High Yield Transcription Kit (Ambion), which yielded 50–100 ug of amplified RNA (aRNA) from each sample.
|
| Label |
Cy5
|
| Label protocol |
2.5 ug aliquots of aRNA from tumor cells or normal cells were annealed with random primer for 10 min at 70oC. They were labeled in a 50ul volume by reverse transcription reaction for 2 hours at 37oC using Superscript II reverse transcriptase (Invitrogen) with 1mM Cy5-dCTP for tumor cells or Cy3-dCTP for normal cells (Amersham) and 25mM each dATP, dGTP, and dTTP. The labeling reaction was stopped by adding 6ul 2.5 N NaOH and incubation for 30 min at 65oC and neutralized by 20ul 1M Tris-HCl (pH7.4) and 7ul 2.5N HCl. The labeled cDNAs were purified by QIAquick PCR Purification Kit (QIAGEN).
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| |
|
| Channel 2 |
| Source name |
Universal control of normal prostatic epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
Mixture of normal prostatic epithelial cells from five patients undergoing radical cystectomy for bladder cancer or suprapubic prostatectomy for benign prostatic hyperplasia.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100,00-200,000 tumor cells or normal cells were isolated selectively using the EZ cut system with a pulsed ultraviolet narrow beam-focus laser (SL Microtest GmbH, Germany) in accordance with the manufacturer’s protocols. Total RNAs were isolated from captured cells with QIAGNE RNAeasy Kit (QIAGEN). After DNAse I treatment, total RNAs were subjected to two rounds of T7-based amplification using Ampliscribe T7 Transcription Kit (Epicentre Technologies), or MEGAscript High Yield Transcription Kit (Ambion), which yielded 50–100 ug of amplified RNA (aRNA) from each sample.
|
| Label |
Cy3
|
| Label protocol |
2.5 ug aliquots of aRNA from tumor cells or normal cells were annealed with random primer for 10 min at 70oC. They were labeled in a 50ul volume by reverse transcription reaction for 2 hours at 37oC using Superscript II reverse transcriptase (Invitrogen) with 1mM Cy5-dCTP for tumor cells or Cy3-dCTP for normal cells (Amersham) and 25mM each dATP, dGTP, and dTTP. The labeling reaction was stopped by adding 6ul 2.5 N NaOH and incubation for 30 min at 65oC and neutralized by 20ul 1M Tris-HCl (pH7.4) and 7ul 2.5N HCl. The labeled cDNAs were purified by QIAquick PCR Purification Kit (QIAGEN).
|
| |
|
| |
| Hybridization protocol |
Labeled cDNAs were mixed with microarray hybridization solution version 2 (Amersham) and formamide (Sigma) to a final concentration of 50%. After hybridization for 14-16 hours at 42 oC in Automated Slide Processor, the slides were washed in 2xSSC and 1% SDS for 10min at 55 oC, washed in 0.2xSSC and 0.1% SDS for 10min at 55 oC, and washed in 0.1xSSC for 1 min at room temperature in Automated Slide Processor.
|
| Scan protocol |
Microarray slides were scanned using the Array Scanner Generation III (Amersham) after hybridization.
|
| Description |
Human hormone-refractory prostate cancer cells were microdissected from clinical frozen sample and after two-round T7-based amplification, Cy5-labeled cDNA was hybridized competitively on cDNA microarrays with Cy3-labeled cDNA of control mixture of amplified RNA from normal prostate epithelial cells that were also microdissected.
|
| Data processing |
Images were gridded and the fluorescent intensity of each spot (Cy5/Cy3) was evaluated photometrically by the ArrayVision computer program (Imaging Research, Inc., St. Catharines, Ontario, Canada) . The fluorescent intensities of Cy5 (tumor) and Cy3 (control) for each target spot were adjusted so that the mean Cy3/Cy5 ratio of the 52 housekeeping genes was equal to one. Because data derived from low signal intensities are less reliable, we determined a cut-off value on each slide and excluded genes from further analysis when both Cy3 and Cy5 dyes yielded signal intensities lower than the cut-off value. For other genes, we calculated the Cy5/Cy3 ratio using the raw data of each sample.
|
| |
|
| Submission date |
Jan 17, 2007 |
| Last update date |
May 02, 2007 |
| Contact name |
Hidewaki Nakaagwa |
| E-mail(s) |
[email protected]
|
| Phone |
+81-3-5449-5375
|
| Fax |
+81-3-5449-5124
|
| Organization name |
The University of Tokyo
|
| Department |
Institute of Medical Science
|
| Lab |
Laboratory of Molecular Medicine
|
| Street address |
4-6-1 Shirokanedai, Minato-ku,
|
| City |
Tokyo |
| State/province |
Tokyo |
| ZIP/Postal code |
108-8639 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4747 |
| Series (1) |
| GSE6811 |
Molecular Features of Hormone-Refractory Prostate Cancer Cells by Genome-wide Gene-expression Profiles |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
the transformed log2 (Cy5_SIG/Cy3_SIG= cancer/normal) ratio |
| RATIO |
the ratio of Cy5_SIG (cancer cell) /Cy3_SIG (normal cell) |
| Cy3_SIG |
the normalized signal of Cy3 (normal control) |
| Cy5_SIG |
the normalized signal of Cy5 (cancer cell) |
| Cy3_CUTOFF |
the cutoff value of Cy3 on each slide, calculated according to the background fluctuation |
| Cy5_CUTOFF |
the cutoff value of Cy5 on each slide, calculated according to the background fluctuation |
