|
| Status |
Public on Apr 01, 2014 |
| Title |
HT-29_exo_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HT-29 cells, exosomal RNA, replicate-2
|
| Organism |
Homo sapiens |
| Characteristics |
microrna source: exosomes
biological source of exosomes or cells: conditioned medium from colon cancer cells
cell type: epithelial
cell line: HT-29
disease state: colorectal adenocarcinoma
|
| Treatment protocol |
Conditioned medium from cell-cultured 100 mm dish (1x10^7 cells) were collected. Exosomes fraction were prepared by step-wise centrifugation-ultracentrifugation method.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Exosome fraction was mixed with Trizol-LS reagent (Invitrogen), and aqueous phase was collected by adding chloroform. After addition of ethanol to the aqueous phase, it was placed on to an RNeasy mini spin column (Qiagen) for the purification of total RNAs.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled miRNA was prepared from total RNA using the miRNA Complete Labeling and Hyb Kit (Agilent) according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
Cy3-labelled miRNA was incubated at 100°C for 5 minutes in a reaction volume of 45uL containing 2x Hi-RPM Hybridization Buffer and 10x Gene Expression blocking agent following the manufacturers instructions and cooled down in ice-cold water for 5 min. Reaction mixture hybridized to Agilent Human miRNA V3 Microarray (G4470C) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green ).
|
| Description |
MicroRNA profile in exosomes of conditioned medium
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol miRNA_105_Dec08 and Grid: 021827_D_F_20081129) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Aug 02, 2012 |
| Last update date |
Apr 01, 2014 |
| Contact name |
Naoto Tsuchiya |
| Organization name |
National Cancer Center Reasearch Institute
|
| Lab |
Genome Biology
|
| Street address |
Tsukiji 5-1-1
|
| City |
Chuo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
104-0045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14767 |
| Series (2) |
| GSE39832 |
Profile analysis of endogenous and exosomal microRNAs in human colon cancer cell lines-2 |
| GSE40247 |
Profiling of endogenous and exosomal microRNAs in colon cancer |
|
