|
| Status |
Public on Sep 27, 2014 |
| Title |
2.5mM_11d_Shoot |
| Sample type |
RNA |
| |
|
| Source name |
Nitrate treatment
|
| Organism |
Zea mays |
| Characteristics |
cultivar: Gaspe Flint
treatment: Nitrate
mm no3-: 2.5
tissue: Shoot
harvest day: 11
replicate: consists of 3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Dwarf maize (Zea mays L. var Gaspe Flint) were grown in hydroponic systems as described previously (Garnett et al., 2013). Plants were sampled between 5 and 7 h after the start of the light period (06:00). The whole root and the youngest fully emerged leaf blade were excised, snap frozen in liquid N and stored at -80°C. Total RNA was extracted from frozen tissue (Chomczynski, 1993), and 10 µg aliquots were prepared for microarray analysis. RNA integrity was checked on a 1.2% (w/v) agarose gel. Three biological replicates were analysed in all experiments.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was treated with DNase-I followed by polyA RNA isolation (Illustra mRNA Purification Kit, GE Biosciences) for all samples. The total RNA and polyA RNA samples were visualized and quantified on Agilent’s Bioanalyzer 2100 to check for degradation and to determine final concentration. Each mRNA sample was made into double stranded DNA, amplified by an in-vitro transcription reaction and labelled with Cy3 fluorescent dye using Agilent’s Low RNA Input Fluorescent Linear Amplification Kit. The cRNA product was purified with Agencourt’s RNAClean Kit that utilizes SPRI (Solid Phase Reversible Immobilization) paramagnetic bead-based technology.
|
| |
|
| Hybridization protocol |
Overnight hybridizations were performed with equal amounts of labelled cRNA to a custom 4x44K Maize Oligo Microarray from Agilent Technologies (Palo Alto, CA) according to Agilent’s One-Color Microarray-Based Gene Expression Analysis protocol. After hybridization, the microarray slides were washed.
|
| Scan protocol |
The microarray slides were immediately scanned with Agilent’s G2505C DNA Microarray Scanner. The images were visually inspected for image artefacts and feature intensities were extracted, filtered, and normalized with Agilent’s Feature Extraction Software (v10.5.1.1). Further quality control and downstream analysis was performed using data analysis tools in GeneData Analyst (v2.2.2).
|
| Description |
Double stranded DNA was amplified by an in-vitro transcription reaction and labeled with Cy3 fluorescent dye. cRNA was hybridized to a custom 4x44K Maize Oligo Microarray.
|
| Data processing |
Cy3 median signal intensities were imported into R for further processing, omitting 4825 probes with no and very low fluorescent signals. The intensity values were log2 transformed and quantile normalized. Differently expressed genes were identified by the moderated t-statistic implemented in the LIMMA package (Smyth 2004). P-values were adjusted employing the method by Benjamini & Hochberg (1995) to control the false discovery rate (FDR). Genes were considered differentially expressed between the two conditions when their adjusted p-values were less or equal to 0.01.
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| |
|
| Submission date |
Sep 26, 2014 |
| Last update date |
Sep 27, 2014 |
| Contact name |
Darren Plett |
| E-mail(s) |
darren.plett@acpfg.com.au
|
| Phone |
+61 8 8313 1499
|
| Organization name |
University of Adelaide
|
| Department |
ACPFG
|
| Street address |
Hartley Grove Road
|
| City |
Urrbrae |
| State/province |
South Australia |
| ZIP/Postal code |
5064 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19236 |
| Series (1) |
| GSE61820 |
Developmental gene expression in Gaspe in response to nitrate |
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