|
| Status |
Public on Dec 12, 2009 |
| Title |
WT vs Hypomethylated Forebrain Gene Expression |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
dorsal cortex of P5 Emx1-cre Dnmt1 mutant mice
|
| Organism |
Mus musculus |
| Characteristics |
dorsal cortex of P5 Emx cre Dnmt1 mutant mice
(Back born strain; C57BL/6)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extracted and cleaned with Qiagen RNeasy minElute column and tested the quality of the RNA on a NanoChip (Agilent).
|
| Label |
Cy5
|
| Label protocol |
We converted the RNA into cDNA and then the cDNA into cRNA using the Agilent Low RNA Input Linear Amplification Kit (Agilent). We using a Nanodrop (Nanodrop) to quantify the labeled cRNA. Cy5 Labelled with Agilent Low Input Linear Amplification Kit
|
| |
|
| Channel 2 |
| Source name |
dorsal cortex of P5 WT mice
|
| Organism |
Mus musculus |
| Characteristics |
dorsal cortex of P5 WT mice
(Back born strain; C57BL/6)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extracted and cleaned with Qiagen RNeasy minElute column and tested the quality of the RNA on a NanoChip (Agilent).
|
| Label |
Cy3
|
| Label protocol |
We converted the RNA into cDNA and then the cDNA into cRNA using the Agilent Low RNA Input Linear Amplification Kit (Agilent). We using a Nanodrop (Nanodrop) to quantify the labeled cRNA. Cy3 Labelled with Agilent Low Input Linear Amplification Kit.
|
| |
|
| |
| Hybridization protocol |
1ug of RNA for each channel was labelled using Agilent Low Input Linear Amplification protocol. Labelled cRNA mixed with Agilent 10x Control targets and Agilent 25x Fragmentation Buffer. This was incubated at 37C for 30 min and the reaction was stopped by the addition of 2x Agilent hybridization buffer. This mix was hybridized to arrays for 17 hours at 65C at 4rpms. Arrays were washed as per Agilent protocol and scanned immediately to prevent ozone degradation.
|
| Scan protocol |
The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (version 8.5)
|
| Description |
Using the cre-loxP binary gene deletion strategy, we crossed female mice homozygous for the Dnmt1 conditional allele (Dnmt12lox (Fan et al., 2001; Jackson-Grusby et al., 2001)) with male mice carrying the Emx1-cre insertion (Emx1-cre, kindly provided by Dr. S. Itohara at RIKEN Brain Research Institute, Japan. See Iwasato et al., 2000) to generate both control, heterozygous, and mutant offspring in expected Mendelian ratios.
RNA samples were extracted from the dorsal cortex of WT and mutant mice at the postnatal day (P) 5.
|
| Data processing |
Arrays were processed using Agilent Feature Extraction (Version 8.5). Log 10 ratio were calculated from processed Cy5/ processed Cy3 signals. Replicates 1 - 3 were labeled - mutant mutant with Cy5 and WT with Cy3.
|
| |
|
| Submission date |
Dec 12, 2008 |
| Last update date |
Nov 15, 2012 |
| Contact name |
Masakazu Namihira |
| E-mail(s) |
namihira@bs.naist.jp
|
| Organization name |
UCLA
|
| Department |
Human Genetics
|
| Lab |
Guoping Fan Lab
|
| Street address |
695 Charles E. Young Dr.
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE14216 |
WT vs Hypomethylated Forebrain Gene Expression |
|
