|
| Status |
Public on Jun 08, 2014 |
| Title |
FaDu_mir10b_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FaDu_mir10b
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: FaDu
cell type: squamous cell carcinoma
genotype/variation: mir10b
|
| Treatment protocol |
SCC25 and FaDu cell lines were grown in a Dulbecco´s Modified Eagle´s medium/Nutrient Mixture F-12 Ham (DMEM/F12) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Keratinocytes were plated on a support layer, called feeder-layer, composed of murine fibroblasts of the type 3T3-Swiss albino (ATCC, catalog number CCL-92), which were irradiated (60 Gy), and maintained in an incubator at 37°C, in a humidified atmosphere containing 5% CO2 and grown as previously described. For transfection, we used the siPORT NeoFx reagent (Ambion) following the manufacturer protocol. For up-regulation, Ambion Pre-miR™ miRNA Precursor Molecule (miR-10b and miR-196a) was used, with Ambion's Pre-miR negative control #1. Successful up-regulation was achieved with 50nM final Pre-miR miRNA Precursor concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cell culture using mirVana miRNA Isolation Kit (Ambion Inc.) in compliance with the manufacturer’s protocol. RNA integrity and concentration were assessed using the RNA 6000 Nano Assay kit with Agilent 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 0.2 ug RNA using the One-Color or Two-Color Low RNA Input Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoVue (GE Healthcare) Spectrophotometer. For two-color labeling samples were labeled with Cy5 and a reference RNA with Cy3(Universal Human Reference RNA, Agilent)
|
| |
|
| Channel 2 |
| Source name |
Universal Human Reference RNA, Agilent
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: reference RNA
|
| Treatment protocol |
SCC25 and FaDu cell lines were grown in a Dulbecco´s Modified Eagle´s medium/Nutrient Mixture F-12 Ham (DMEM/F12) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Keratinocytes were plated on a support layer, called feeder-layer, composed of murine fibroblasts of the type 3T3-Swiss albino (ATCC, catalog number CCL-92), which were irradiated (60 Gy), and maintained in an incubator at 37°C, in a humidified atmosphere containing 5% CO2 and grown as previously described. For transfection, we used the siPORT NeoFx reagent (Ambion) following the manufacturer protocol. For up-regulation, Ambion Pre-miR™ miRNA Precursor Molecule (miR-10b and miR-196a) was used, with Ambion's Pre-miR negative control #1. Successful up-regulation was achieved with 50nM final Pre-miR miRNA Precursor concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cell culture using mirVana miRNA Isolation Kit (Ambion Inc.) in compliance with the manufacturer’s protocol. RNA integrity and concentration were assessed using the RNA 6000 Nano Assay kit with Agilent 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 0.2 ug RNA using the One-Color or Two-Color Low RNA Input Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoVue (GE Healthcare) Spectrophotometer. For two-color labeling samples were labeled with Cy5 and a reference RNA with Cy3(Universal Human Reference RNA, Agilent)
|
| |
|
| |
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (and Cy5-labelled cRNA, if applicable) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on theGenePix Scanner using settings suggested by Agilent
|
| Description |
Gene Expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v.5 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jun 13, 2013 |
| Last update date |
Oct 04, 2023 |
| Contact name |
Patricia Severino |
| E-mail(s) |
patricia.severino@einstein.br
|
| Phone |
5511984661282
|
| Organization name |
Hospital Israelita Albert Einstein
|
| Department |
Albert Einstein Research and Education Institute
|
| Street address |
Rua Comendador Elias Jafet, 755
|
| City |
Sao Paulo |
| State/province |
Sao Paulo |
| ZIP/Postal code |
05653-000 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL10332 |
| Series (1) |
| GSE31277 |
MicroRNA expression profile in Head and Neck Cancer: HOX-cluster embedded microRNA-196 and microRNA-10b dysregulation is implicated in cell proliferation |
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