|
| Status |
Public on Sep 30, 2014 |
| Title |
Neural Tube-mutant-rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mutant embryo#2, neural tube
|
| Organism |
Mus musculus |
| Characteristics |
developmental stage: E10.5 embryo
strain: C57BL/6
genotype/variation: fl/fl; cre/o
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared from laser captured material by following the manufacturer's instructions in the Arcturus PicoPure RNA Isolation kit from Life Technologies. Total RNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. Total RNA quality was then determined by Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer’s recommendations.
|
| Label |
Cy5
|
| Label protocol |
cDNAs were chemically labeled with Kreatech ULS labeling kit (Kreatech Diagnostics). Per reaction, 2.5ug of DNA (+water=16ul) was mixed with Kreatech 10x labeling buffer (2ul) and Kreatech cy5-ULS (2ul). The reactions were incubated at 85C for 15 minutes in the dark and placed on ice for 3 minutes. Labeled DNAs were purified with QIAquick PCR purification columns (Qiagen Sciences). Labeled DNAs were quantitated on a Nanodrop spectrophotometer.
|
| |
|
| Hybridization protocol |
2ug of each labeled DNA (+water=17ul) was suspended in Agilent 2X Gene Expression hyb buffer (25ul), Agilent 10X Blocking agent (5ul), and Kreatech Kreablock (3ul). The hybridization solutions were applied to Agilent Mouse8x60K microarrays. Hybridization was carried out at 65C for 20 hours. Washing procedures were carried out according to Agilent gene expression protocols.
|
| Scan protocol |
Slides were scanned on an Agilent SureScan microarray scanner to detect Cy5 fluorescence. Gridding and analysis of images was performed using Agilent Feature Extraction v11.0.1.1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09_red_only and Grid: 028005_D_F_20100422) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Nov 05, 2012 |
| Last update date |
Oct 01, 2014 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
kkroll@wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Developmental Biology
|
| Lab |
Kristen Kroll
|
| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE42052 |
Analysis of conditional loss of Geminin in neural tube and paraxial tissues at E10.5 by laser capture microdissection |
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