|
| Status |
Public on Sep 01, 2011 |
| Title |
root low N biological replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
maize root at 0.04mM nitrate culture for 15 days
|
| Organism |
Zea mays subsp. mays |
| Characteristics |
tissue: root tips
genotype: Ye478, an inbred line
age: roots around 20 days after germination
treatment: 0.04 mM nitrate
|
| Treatment protocol |
Two nitrate concentrations were tested: 4 mM, which represented an optimal nitrate condition (high N) and 0.04 mM, which represented low-nitrate availability (low N) with Ca (NO3)2•4H2O used as the nitrate source. Ca was compensated to 2 mM at low N with CaCl2 . The other nutrients in solution were (in mmol L-1): 0.75 K2SO4, 0.1 KCl, 0.25 KH2PO4, 0.65 MgSO4.7H2O, and 0.2 EDTA-Fe, and in μmol L-1, 1.0 MnSO4.H2O, 1.0 ZnSO4.7H2O, 0.1 CuSO4.5H2O, and 0.005 (NH4)6Mo7O24.4H2O. Air was continuously pumped through the solution (pH 6.0) that was changed every 2 days. For identification of chronic nitrate regulated miRNAs, seedlings were sampled at 15 days for RNA extraction. For transient expression pattern of miRNAs from high to low N, the 15-day-old seedlings were transferred from high N to low N conditions for hours.
|
| Growth protocol |
Seeds of Ye478 were sterilized with 10% (v/v) H2O2 for 30 min, washed with distilled water, soaked in saturated CaSO4 for 6 h, and then germinated at 28 °C for 2 d in the dark between two layers of filter paper moistened with saturated CaSO4. Seeds 1-2 cm germ were transferred to coarse silica sand to grow at 28 °C /22 °C during the 14/10 h light / dark cycle. Uniform seedlings with two visible leaves were selected. The residual endosperms of the seedlings were discarded. Plants were planted in a glass beaker. The outside of the glass beakers were covered with a black sheet to ensure that the roots were kept in complete darkness. Glass beakers each containing ten seedlings were maintained in an illumination chamber.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
20×Eukaryotic Hybridization Controls (GeneChip○R Eukaryotic Hybridization Control Kit, Affymetrix) were incubated at 65 °C for 5 min. 21.5 μl biotin-labeled RNA was suspended in 78.5 μl hybridization solution containing 2×Hybridization Mix, deionized formamide, DMSO, 20×Eukaryotic Hybridization Controls, 3 nM Control Oligonucleotide B2 and nuclease-free water, which was incubated at 99 °C for 5 min, followed by 45 °C for 5 min. The hybridization was performed at 48 °C for 16 h. The arrays were then washed and stained with GeneChip○R Hybridization Wash and Stain Kit (Affymetrix, Inc.)
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000.
|
| Data processing |
The Affymetrix○C miRNA QC Tool software (Affymetrix, Inc.) was used for data summarization, normalization, and quality control. The miRNAs with P<0.05(q<0.001) and fold changes >2.0 or <0.5 were defined as differentially expressed. Three biological replicates were used in all chip hybridization experiments.
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| |
|
| Submission date |
Aug 05, 2011 |
| Last update date |
Sep 01, 2011 |
| Contact name |
Chuanxiao Xie |
| E-mail(s) |
cxxie@caas.net.cn
|
| Organization name |
Chinese Academy of Agricultural Sciences
|
| Department |
Institute of Crop Science
|
| Lab |
Center for maize study
|
| Street address |
Zhong-guan-cun South St. No12
|
| City |
Beijing |
| ZIP/Postal code |
100081 |
| Country |
China |
| |
|
| Platform ID |
GPL8786 |
| Series (2) |
| GSE31231 |
Expression data from low nitrate culture in root in maize |
| GSE31492 |
miRNA expression data from low nitrate cultures in maize |
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