|
| Status |
Public on Mar 12, 2010 |
| Title |
fibroblasts_miRNA transfected_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
AG01522 normal human fibroblasts, miRNA-transfected
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: cultured fibroblasts
cell line: AG01522
supplier: Coriell
passage: 20
transfection: miRNA
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=AG01522
|
| Treatment protocol |
Typically, for a 60 mm plate, 500 µl of optiMEM (Invitrogen) was mixed with 2.5 µl of TransIT-siQUEST (Mirus) and incubated for 10 min. The small RNA fraction (800 ng in ~15 µl) was added to this mixture and incubated for 10 more min. This mixture was placed on the bottom of a 60 mm plate and immediately 500,000 cells in 3.5 ml of complete media were added followed by incubation of the cells for 38 h. The media was changed after 24 h. Control cells were mock-transfected using the same conditions without the addition of RNA.
|
| Growth protocol |
Human fibroblasts (AG01522) were received as cell cultures from Coriell at passage (PDL) 16 and expanded until PDL 20 by culturing in EEMEM (Invitrogen) supplemented with 15% FBS (ATCC).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Ribopure kit (Applied Biosystems), following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
For array analysis, 0.3 µg RNA was used to produce Cyanine-3 (Cy3)-labeled cRNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent).
|
| |
|
| Hybridization protocol |
Following the manufacturer’s recommendations, 1.5 µg labeled cRNA (>9 pmol Cy3 per µg cRNA) was fragmented and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) using the Gene Expression Hybridization Kit.
|
| Scan protocol |
Microarrays with labeled cRNA were scanned at 5 µm resolution with an Agilent DNA Microarray Scanner (G2505B).
|
| Description |
Gene expression after transfection with endogenous small RNA fraction.
mir_5_72_1
|
| Data processing |
Default parameters of Feature Extraction v. 10.5 software (Agilent) were used for image analysis, data extraction, background correction, and flagging of non-uniform features. Background-corrected intensities were log2-transformed and median-normalized using BRB-ArrayTools v. 3.8.0. Non-uniform outliers or features not significantly above background intensity in 25% or more of the hybridizations were removed, leaving a set of 5800 features that were used for subsequent analyses.
|
| |
|
| Submission date |
Mar 11, 2010 |
| Last update date |
Mar 22, 2010 |
| Contact name |
Thomas Templin |
| E-mail(s) |
tt2308@columbia.edu
|
| Phone |
212-305-5661
|
| Fax |
212-305-3229
|
| URL |
http://www.crr-cu.org/templin.htm
|
| Organization name |
Columbia University Medical Center
|
| Street address |
630 West 168th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE20755 |
Identification and analysis of miRNA target genes in a cell system |
|
