|
| Status |
Public on Jul 16, 2012 |
| Title |
RC_KI04007 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
clear cell renal cell carcinoma (ccRCC)
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
age: 73
pt: 1b
grade: G3>G2
tissue origin: surgically-resected renal cell carcinoma
cell type: Clear cell renal cell carcinoma cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Celar cell renal cell carcinoma cells that were microdissected from tissue samples of surgically-resected renal cell carcinoma. 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 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).
|
| |
|
| Channel 2 |
| Source name |
Universal control of normal renal cortex
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: A mixture of normal renal cortex cells in kidney tissues from 11 patients
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Celar cell renal cell carcinoma cells that were microdissected from tissue samples of surgically-resected renal cell carcinoma. 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 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 55C, washed in 0.2xSSC and 0.1% SDS for 10min at 55C, 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.
|
| 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. Experiments were performed using 6 sets of slides (slide set 1-6 corresponding to ID_REF 1-27648).
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| |
|
| Submission date |
Jul 15, 2012 |
| Last update date |
Jul 16, 2012 |
| Contact name |
Toyomasa Katagiri |
| E-mail(s) |
tkatagi@genome.tokushima-u.ac.jp
|
| Phone |
81-88-633-9477
|
| Fax |
81-88-633-7986
|
| Organization name |
The University of Tokushima
|
| Street address |
3-18-15 Kuramoto-cho
|
| City |
Tokushima |
| State/province |
Tokushima |
| ZIP/Postal code |
770-8503 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4747 |
| Series (1) |
| GSE39364 |
Genome-wide gene expression profiles of clear cell renal cell carcinoma; Identification of molecular targets for treatment of renal cell carcinoma |
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