|
| Status |
Public on Sep 01, 2013 |
| Title |
Coleoptilar_node_WT_0d_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Coeloptilar node
|
| Organism |
Zea mays |
| Characteristics |
tissue: Coeloptilar node
genotype/variation: wild type
developmental stage: day 0
|
| Treatment protocol |
For Agilent microarray profiling, coleoptilar nodes of wild-type and rtcs seedlings 0 (t0), 2 (t2), and 4 (t4) days after the emergence of these structures were used. Each coleoptilar node RNA extract (biological replicate) was obtained from a pool of approximately 10 coleoptilar nodes collected from seedlings of a single laboratory AC. For each genotype/stage combination four biological replicates were hybridized.
|
| Growth protocol |
Seedlings were grown in a plant growth chamber at 26°C, with a 16 h light, 8 h dark program at 60% humidity. Coleoptilar nodes were manually dissected from seedlings and immediately frozen in liquid nitrogen after isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from approximately 500 mg of frozen coleoptilar nodes per biological replicate via the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For all samples, polyA RNA was isolated using the “Illustra mRNA Purification Kit” (GE Biosciences, Piscataway, NJ) and analyzed on Agilent’s (Colorado Springs, CO) Bioanalyzer to check for degradation and to determine concentration.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labelled cRNA was synthesized from 100 ng of polyA RNA using the “Quick Amp Labeling Kit One Color” (Agilent, Santa Clara, CA, USA) which generates fluorescent complimentary RNA (cRNA) by simultaneously amplifying target material and incorporating cyanine 3-labeled dCTP via T7 polymerase.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNAs were hybridized in “one-color mode” using Agilent´s “Gene Expression Hybridization Kit” for 17 h at 65°C according to the manufacturer´s protocol. After hybridization microarrays were washed twice with Agilent´s “Gene Expression Wash Buffer” 1 and 2 for one minute each at room temperature and 37°C, respectively followed by a brief wash in acetonitrile.
|
| Scan protocol |
Scanning of arrays was performed with Agilent´s G2505B DNA Microarray Scanner at 5 micron resolution in the single path scanning mode of the green dye channel at two laser settings (10% and 100% PMT) using Agilent’s “Scan Control software, version A. 7.0.1”.
|
| Description |
chip barcode: 251553810408_1_1
|
| Data processing |
Features were extracted with Agilent's “Feature Extraction 9.5.3 Image Analysis Software” and normalized via the mean intensity feature of the Rosetta Resolver program (Rosetta Biosoftware, Cambridge, MA, USA) after excluding control features and flagged microarray features and trimming the top and bottom 10% of the data. A p-value threshold of 0.01 was applied which was calculated with Agilent's single color error model.
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| |
|
| Submission date |
Nov 18, 2010 |
| Last update date |
Sep 01, 2013 |
| Contact name |
Frank Hochholdinger |
| E-mail(s) |
hochhold@uni-bonn.de
|
| Phone |
0049 228 73 60334
|
| Fax |
0049 228 73 60333
|
| URL |
http://www.lwf.uni-bonn.de/institute/inres/institut/crop-functional-genomics/
|
| Organization name |
University of Bonn
|
| Department |
INRES - Crop Functional Genomics
|
| Lab |
Hochholdinger
|
| Street address |
Katzenburgweg 1a
|
| City |
Bonn |
| State/province |
BW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10837 |
| Series (1) |
| GSE25467 |
Maize coleoptilar node development: WT vs. rtcs |
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