|
| Status |
Public on Oct 26, 2007 |
| Title |
Gene expression changes in human colorectal cell line HCT116 DICER Exon5 knockout cells |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Human colorectal HCT116 DICER Exon5 knockout cells
|
| Organism |
Homo sapiens |
| Characteristics |
Human colorectal HCT116 DICER exon5 knockout cells
|
| Biomaterial provider |
Dr. Bert Vogelstein
|
| Treatment protocol |
There were no specific treatments for cells during growth.
|
| Growth protocol |
Cells were grown in McCoy's 5A medium supplimented with 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated by using TriZol reagent followed by clean-up with RNeasy mini kit plus on-column DNease treatment.
|
| Label |
Cy5
|
| Label protocol |
Sample amplification and labeling procedures are carried out by using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). ). 0.4 microgram of total RNA is reverse transcribed into first strand and second strand cDNA by MMLV-RT using an oligo dT primer (System Biosciences) that incorporates a T7 promoter sequence. The cDNA is then used as a template for in vitro transcription in the presence of T7 RNA polymerase and Cyanine labeled CTPs. The labeled cRNA is purified using RNeasy mini kit (Qiagen) and followed by quantification on both concentrations of cRNA and dye labeled. RNA spike-in controls (Agilent Technologies) are added to RNA samples before amplification and labeling according to manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
Human colorectal HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
Human colorectal HCT116 cells
|
| Biomaterial provider |
Dr. Bert Vogelstein
|
| Treatment protocol |
There were no specific treatments for cells during growth.
|
| Growth protocol |
Cells were grown in McCoy's 5A medium supplimented with 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated by using TriZol reagent followed by clean-up with RNeasy mini kit plus on-column DNease treatment.
|
| Label |
Cy3
|
| Label protocol |
Sample amplification and labeling procedures are carried out by using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). ). 0.4 microgram of total RNA is reverse transcribed into first strand and second strand cDNA by MMLV-RT using an oligo dT primer (System Biosciences) that incorporates a T7 promoter sequence. The cDNA is then used as a template for in vitro transcription in the presence of T7 RNA polymerase and Cyanine labeled CTPs. The labeled cRNA is purified using RNeasy mini kit (Qiagen) and followed by quantification on both concentrations of cRNA and dye labeled. RNA spike-in controls (Agilent Technologies) are added to RNA samples before amplification and labeling according to manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
Fragmented targets are added an Oligo Microarray, assembled into a hybridization chamber (Agilent Technologies) and hybridized at 65ºC for 17 hours in a hybridization oven with rotation. Hybridized microarrays are washed and dried according to the Agilent microarray processing protocol.
|
| Scan protocol |
Microarrays are scanned with Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies for expression microarrays with 100% PMT and 10 um resolutions.
|
| Description |
None
|
| Data processing |
Data are extracted using Feature Extraction Software v8.5 (Agilent Technologies) under default settings for expression microarrays with linear & lowess normalization.
VALUEs represent log10 (test/reference) ratios, after linear and loess normalization.
|
| |
|
| Submission date |
Dec 01, 2006 |
| Last update date |
Apr 26, 2007 |
| Contact name |
Wayne Yu |
| E-mail(s) |
wyu8@jhmi.edu
|
| Phone |
(410)502-7970
|
| Fax |
(410)955-8780
|
| Organization name |
Johns Hopkins University
|
| Department |
Cancer Center
|
| Street address |
417 N. Caroline St.
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL1708 |
| Series (1) |
| GSE6427 |
Gene expression changes in human colorectal cell line HCT116 DICER Exon5 knockout cells |
|
