|
| Status |
Public on Mar 08, 2017 |
| Title |
Normal_T190_N |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Normal Prostate
|
| Organism |
Homo sapiens |
| Characteristics |
sample id: T190
tissue: Normal Prostate
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue and biopsy samples were homogenized (Ultra – TurraxT8, Ika – Werke) and totRNA was isolated following TriReagent (Sigma Aldrich) protocol. TotRNA quality and quantity were checked by means of Agilent 2100 bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) respectively.
|
| Label |
Cy5
|
| Label protocol |
RNA isolated from tissues and from a commercial pool of RNA from organ donor healthy prostates (Becton Dickinson) was amplified by means of the Amino Allyl MessageAmp aRNA Kit (Life Technologies) 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) able to react with the modified RNA. Labeling efficiency and mRNA quantity were checked by means of NanoDrop ND-1000 Spectrophotometer (Thermo Scientific).
|
| |
|
| Channel 2 |
| Source name |
Prostate Commercial Reference
|
| Organism |
Homo sapiens |
| Characteristics |
reference: a commercial pool of RNA from organ donor healthy prostates (Becton Dickinson)
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue and biopsy samples were homogenized (Ultra – TurraxT8, Ika – Werke) and totRNA was isolated following TriReagent (Sigma Aldrich) protocol. TotRNA quality and quantity were checked by means of Agilent 2100 bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) respectively.
|
| Label |
Cy3
|
| Label protocol |
RNA isolated from tissues and from a commercial pool of RNA from organ donor healthy prostates (Becton Dickinson) was amplified by means of the Amino Allyl MessageAmp aRNA Kit (Life Technologies) 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) able to react with the modified RNA. Labeling efficiency and mRNA quantity were checked by means of NanoDrop ND-1000 Spectrophotometer (Thermo Scientific).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of differentially labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays with sequences representing 27,958 Entrez Gene RNAs and 7,419 lincRNAs (Human Gene Expression 8x60K Microarray, 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 following the Agilent wash procedure. For each sample, a dye-swap replicate was performed.
|
| Scan protocol |
Slides were scanned with the dual-laser Agilent scanner G2505C
|
| Description |
Peripheral region of the prostate from Caucasian men without PCa vs Prostate Commercial Reference (normal labelling)
|
| Data processing |
The LIMMA (LInear Models for Microarray Analysis) package was used to analyze gene expression profiles of normal prostates and prostate cancer tissues. Raw intensity values were background subtracted (method = normexp, offset = 50) and normalized using the loess method, for the within array normalizaton, and Aquantile, for the between array normalization. Duplicated probes were averaged. Then, Cy3 and Cy5 channels were separated using the separate channel analysis of two-color data, available within the Limma package.
|
| |
|
| Submission date |
Aug 12, 2014 |
| Last update date |
Mar 08, 2017 |
| Contact name |
Paola Ostano |
| Organization name |
Fondazione Edo ed Elvo Tempia
|
| Street address |
via Malta 3
|
| City |
Biella |
| ZIP/Postal code |
13900 |
| Country |
Italy |
| |
|
| Platform ID |
GPL14550 |
| Series (2) |
| GSE60329 |
Gene expression profiling of prostate cancer (PCa) and normal prostatic samples |
| GSE60371 |
miR-424 induces COP1 silencing and STAT3 activation in prostate cancer: a novel miRNA-dependent axis driving tumor progression |
|
