|
| Status |
Public on Nov 04, 2016 |
| Title |
Maize-GLS-RILs-138_220 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Leaf, Grey leafspot
|
| Organism |
Zea mays |
| Characteristics |
ril: 138
age: 103 DAP
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions, followed by Dnase treatment and Qiagen column purification
|
| Label |
Cy5
|
| Label protocol |
RNA (1μg) was amplified from each of the 100 RIL samples using the Amino Allyl MessageAmpII aRNA Amplification kit (Ambion, Dallas, USA) and labeled with either Cy3 or Cy5 (100 pmol each) according to the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
Leaf, Grey leafspot
|
| Organism |
Zea mays |
| Characteristics |
ril: 220
age: 103 DAP
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions, followed by Dnase treatment and Qiagen column purification
|
| Label |
Cy3
|
| Label protocol |
RNA (1μg) was amplified from each of the 100 RIL samples using the Amino Allyl MessageAmpII aRNA Amplification kit (Ambion, Dallas, USA) and labeled with either Cy3 or Cy5 (100 pmol each) according to the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
Labelled aRNA was hybridized to the Agilent-016047 maize 4×44 K microarrays using the Quadchamber on the Tecan Hybridisation station. The Agilent Two-Color Microarray-Based Gene Expression protocol was followed.
|
| Scan protocol |
The microarrays were scanned using a Tecan LS Re-loaded scanner (Tecan, Mannedorf, Switzerland). Image acquisition was carried out using automatic gain control settings for Cy3 and Cy5. Images from both channels were saved as separate tif files. Spots were flagged as bad if the signal to noise ration was <3 for either the Cy3 or Cy 5 channel.
|
| Description |
Pool of three biological replicates of each RIL line
|
| Data processing |
Cy 5 and Cy3 TIF images for each slide were imported into the Genepix Pro 6.1 software (Molecular Devices, Sunnyvale USA), overlaid on top of each other and saved as a single multitif image. This image was used for all further analysis in Genepix. Four GPR files were exported per slide. Normalization was performed in the R-based package limma, with a weighting of zero for flagged spots. Background correction was performed using the normexp method (offset =50). The loess method was used for normalization within arrays and Aquantile for normalization between arrays. After normalization, 50 datasets of M values (M=log2 Cy5/Cy3) represented the 100 RILs. Back-conversion of the normalized data was required to obtain separate, normalised expression values per reporter for each of the 100 RILs.
|
| |
|
| Submission date |
Dec 21, 2015 |
| Last update date |
Nov 05, 2016 |
| Contact name |
Shane Murray |
| E-mail(s) |
shane.murray@uct.ac.za
|
| Organization name |
University of Cape Town
|
| Department |
Dept of Molecular and Cell Biology
|
| Street address |
University Avenue North
|
| City |
Cape Town |
| ZIP/Postal code |
7700 |
| Country |
South Africa |
| |
|
| Platform ID |
GPL16089 |
| Series (1) |
| GSE76242 |
Microarrays of Maize Recombinant Inbred Lines inoculated with Cercospora zeina, which causes grey leaf spot (GLS) disease |
|
