|
| Status |
Public on Jun 30, 2011 |
| Title |
prostate cancer: 74 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
prostate cancer
|
| Organism |
Homo sapiens |
| Characteristics |
disease: prostate cancer
tag type: BB36
age: 64
stage: T2B
gleason grade: 4-3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All the samples were homogenized (Ultra – TurraxT8, Ika – Werke, Staufen, Germany) and total RNA was isolated by using TriReagent (Sigma Aldrich). TotRNA quality was checked by means of RNA 6000 pico chip assays (Agilent Technologies, Palo Alto, CA) run on the Agilent 2100 bioanalyzer.
|
| Label |
Cy5,Cy3
|
| Label protocol |
RNA isolated from each tissue and from a commercial pool of RNA from organ donor healthy prostates (Becton Dickinson) was amplified by means of the Amino Allyl MessageAmp I aRNA Kit (Ambion) to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis, cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences, Buckinghamshire UK) able to react with the modified RNA. mRNA quality was checked by means of RNA 6000 nano chip assays (Agilent Technologies). At least 5 ug of mRNA for each sample were labeled and purified with columns.
|
| |
|
| Channel 2 |
| Source name |
commercial pool of RNA from organ donor healthy prostates
|
| Organism |
Homo sapiens |
| Characteristics |
reference: commercial pool of RNA from organ donor healthy prostates (Becton Dickinson)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All the samples were homogenized (Ultra – TurraxT8, Ika – Werke, Staufen, Germany) and total RNA was isolated by using TriReagent (Sigma Aldrich). TotRNA quality was checked by means of RNA 6000 pico chip assays (Agilent Technologies, Palo Alto, CA) run on the Agilent 2100 bioanalyzer.
|
| Label |
Cy3,Cy5
|
| Label protocol |
RNA isolated from each tissue and from a commercial pool of RNA from organ donor healthy prostates (Becton Dickinson) was amplified by means of the Amino Allyl MessageAmp I aRNA Kit (Ambion) to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis, cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences, Buckinghamshire UK) able to react with the modified RNA. mRNA quality was checked by means of RNA 6000 nano chip assays (Agilent Technologies). At least 5 ug of mRNA for each sample were labeled and purified with columns.
|
| |
|
| |
| Hybridization protocol |
Equal amounts (0.75 ug) of labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays representing 18K human unique genes and transcripts (Human 1A Glass Oligo Array, Agilent Technologies). All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed with the SSPE wash procedure. For each sample, a dye-swap replicate was performed.
|
| Scan protocol |
Slides were scanned with the dual-laser microarray scanner Agilent G2505B
|
| Description |
technical replicate of 74_NORM
|
| Data processing |
Images were analyzed using Feature Extraction software (Agilent Technologies) version 7.6. Output files containing feature and background intensities and the related statistical parameters for red and green signals were then loaded into the Resolver SE System (Rosetta Biosoftware, Seattle, WA) together with the scan images and the pattern file. Data processing and normalization were performed using the Agilent Human 22k platform-specific error model. Replicated expression profiles were combined to form log10 expression ratios to measure the modulation in the sample compared to the reference.
|
| |
|
| Submission date |
May 15, 2009 |
| Last update date |
Jun 30, 2011 |
| Contact name |
giovanna chiorino |
| E-mail(s) |
giovanna.chiorino@gmail.com
|
| Organization name |
Fondo Edo Tempia
|
| Department |
Cancer Genomics
|
| Street address |
via malta 3
|
| City |
Biella |
| ZIP/Postal code |
13900 |
| Country |
Italy |
| |
|
| Platform ID |
GPL887 |
| Series (1) |
| GSE16120 |
Gene profiling, mutations and expression of epidermal growth factor receptor in androgen-dependent prostate cancer |
|
