|
| Status |
Public on Mar 09, 2017 |
| Title |
RWPE-1_Pre-miR-424_vs_Pre-Ctrl_DS |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RWPE-1 transfected with pre-control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RWPE-1
cell type: noncancerous prostate epithelial cell line
transfection: PRE-CTR
|
| Treatment protocol |
RWPE-1 were transfected with 30nM of Pre-miR-424 or Negative control #1.
|
| Growth protocol |
RWPE-1 were maintained in keratinocyte serum-free growth medium (KSF; Lonza) with specific supplements.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Direct-zol RNA Mini-prep kit (Zymo Research).
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified by means of the Amino Allyl MessageAmp II 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. mRNA amplification was checked by means of Bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences) able to react with the modified RNA. Labeling efficiency and labeled mRNA quantity were checked by means of NanoDrop ND-1000 Spectrophotometer (Thermo Scientific).
|
| |
|
| Channel 2 |
| Source name |
RWPE-1 transfected with pre-miR-424
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RWPE-1
cell type: noncancerous prostate epithelial cell line
transfection: PRE-miR-424
|
| Treatment protocol |
RWPE-1 were transfected with 30nM of Pre-miR-424 or Negative control #1.
|
| Growth protocol |
RWPE-1 were maintained in keratinocyte serum-free growth medium (KSF; Lonza) with specific supplements.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Direct-zol RNA Mini-prep kit (Zymo Research).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified by means of the Amino Allyl MessageAmp II 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. mRNA amplification was checked by means of Bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences) able to react with the modified RNA. Labeling efficiency and labeled 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 (Agilent-028004 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 |
RWPE-1 with pre-miR-424 vs. control, dye-swap labelling.
|
| Data processing |
The LIMMA (LInear Models for Microarray Analysis) package was used to analyze gene expression profiles of RWPE-1 following pre-miR-424 transfection versus control. 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.
|
| |
|
| Submission date |
Aug 07, 2014 |
| Last update date |
Mar 09, 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) |
| GSE60201 |
Gene expression profiling of noncancerous prostate epithelial cells (RWPE-1) following pre-miR-424 transfection |
| GSE60371 |
miR-424 induces COP1 silencing and STAT3 activation in prostate cancer: a novel miRNA-dependent axis driving tumor progression |
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