|
| Status |
Public on Aug 24, 2012 |
| Title |
Prostate_cancer_vs._benign_6082-03 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
6082-03_LCM_normal_adjacent
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 66
tissue: prostate needle biopsy tissue frozen in OCT blocks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from laser captured epithelium using a Picopure RNA isolation kit according to the manufacturer's instructions (Molecular Devices). Extracted RNA underwent two rounds of amplification by a linear T7-RNA polymerase method developed by Van Gelder et al. (31) using a MessageAMP aRNA kit (Applied Biosystems/Ambion).
|
| Label |
Cy5
|
| Label protocol |
cDNA was separately synthesized from 3 μg of aRNA amplified from benign and neoplastic epithelium in a reaction volume of 30 μL containing 5 μg random hexamers (Invitrogen); 0.5 mmol/L each of dATP, dCTP, and dGTP; 0.3 mmol/L dUTP; 0.2 mmol/L aminoallyl-dUTP (Sigma); and 380 units of SuperScript II reverse transcriptase (Invitrogen). Reactants were incubated at 42°C for 120 min followed by the hydrolysis of RNA in 0.2N NaOH at 65°C for 15 min and neutralized by Tris-Cl (pH 7.4). cDNA was cleaned using a Microcon 30 filter (Millipore) to remove primers and salts followed by coupling to Cy3 and Cy5 fluorescent dyes (Amersham Biosciences). To eliminate potential dye bias, we randomly alternated Cy3 and Cy5 labeling to neoplastic and benign epithelium across 31 samples.
|
| |
|
| Channel 2 |
| Source name |
6082-03_LCM_GL3+3
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 66
tissue: prostate needle biopsy tissue frozen in OCT blocks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from laser captured epithelium using a Picopure RNA isolation kit according to the manufacturer's instructions (Molecular Devices). Extracted RNA underwent two rounds of amplification by a linear T7-RNA polymerase method developed by Van Gelder et al. (31) using a MessageAMP aRNA kit (Applied Biosystems/Ambion).
|
| Label |
Cy3
|
| Label protocol |
cDNA was separately synthesized from 3 μg of aRNA amplified from benign and neoplastic epithelium in a reaction volume of 30 μL containing 5 μg random hexamers (Invitrogen); 0.5 mmol/L each of dATP, dCTP, and dGTP; 0.3 mmol/L dUTP; 0.2 mmol/L aminoallyl-dUTP (Sigma); and 380 units of SuperScript II reverse transcriptase (Invitrogen). Reactants were incubated at 42°C for 120 min followed by the hydrolysis of RNA in 0.2N NaOH at 65°C for 15 min and neutralized by Tris-Cl (pH 7.4). cDNA was cleaned using a Microcon 30 filter (Millipore) to remove primers and salts followed by coupling to Cy3 and Cy5 fluorescent dyes (Amersham Biosciences). To eliminate potential dye bias, we randomly alternated Cy3 and Cy5 labeling to neoplastic and benign epithelium across 31 samples.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA probes were hybridized in a head-to-head fashion, normal versus neoplastic from the same individual, simultaneously to custom-made microarrays composed of 6,760 clones derived from the Prostate Expression Database, a public sequence repository of expressed sequence tag data derived from human prostate cDNA libraries (32). Microarrays were constructed as described previously with each cDNA represented twice per array (28). Following probe addition, coverslips were applied and microarrays were incubated at 63°C for 16 h in a humid hybridization chamber. Unbound probe was removed through washes with gradients of SSC buffer.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA). The image intensity data were gridded and extracted using GenePix Pro 4.1 software.
|
| Description |
Prostate_cancer_vs._normal_6082-03
|
| Data processing |
GenePix Pro 4.1 software was used to grid and extract image intensity data. Spots of poor quality as determined by visual inspection were removed from further analysis. Normalization of the Cy3 and Cy5 fluorescent signal on each array was done using Silicon Genetics GeneSpring 7.3 software (Agilent Technologies). A print tip-specific Lowess curve was fit to the background-subtracted log intensity versus log ratio plot and 20.0% of the data were used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10, then 10 was used instead.
|
| |
|
| Submission date |
Aug 24, 2012 |
| Last update date |
Aug 24, 2012 |
| Contact name |
Ilsa Coleman |
| E-mail(s) |
icoleman@fredhutch.org
|
| Phone |
206-667-1703
|
| Organization name |
Fred Hutchinson Cancer Center
|
| Department |
Human Biology
|
| Lab |
Peter Nelson
|
| Street address |
1100 Fairview Ave N, E2-112
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL4767 |
| Series (1) |
| GSE40373 |
Prostate Cancer–Associated Gene Expression Alterations Determined from Needle Biopsies |
|
