|
| Status |
Public on Feb 24, 2010 |
| Title |
Brassica napus_root_Fe-starvation (3 weeks) vs. full nutrition |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brassica napus, root, 3 weeks without iron
|
| Organism |
Brassica napus |
| Characteristics |
cultivar: Drakkar
age: 10 weeks
tissue: root
developmental stage: flowering
cultivation type: hydroponically
treatment: iron starvation
|
| Treatment protocol |
Iron starvation was applied for three weeks before flowering started by changing to medium without iron. Here, the 0.04 mM Fe-EDTA was omitted from the medium.
|
| Growth protocol |
For hydroponic growth, Brassica napus seeds were germinated on wet filter paper for 1 week. Germ buds were transferred to plastic boxes containing nutrient medium for 10 weeks. Nutrient medium: 0.6 mM NH4NO3, 1 mM Ca(NO3)2*4H2O, 0.04 mM Fe-EDTA, 0.5 mM K2HPO4, 0.5 mM K2SO4, 0.4 mM Mg(NO3)2*6H2O. Micro nutrients added: 0.8 µM ZnSO4*7H2O, 9 µM MnCl2*4H2O, 0.1 µM Na2MoO4*2H2O, 23 µM H3BO3, 0.3 µM CuSO4*5H2O. The pH was adjusted to 4.7 with 37 % HCl. Nutrient solutions were changed after 4 weeks, and then renewed once a week. After 5 to 6 weeks, media were constantly aerated by an aquarium air pump.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 100 mg frozen material of root tissue was isolated by using Trizol reagent (Invitrogen) according to manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Microarray assay was performed using the service provider LC Sciences (Houston, TX). The assay started from 2 to 5 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (from Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry.
|
| |
|
| Channel 2 |
| Source name |
Brassica napus, root, full nutrition
|
| Organism |
Brassica napus |
| Characteristics |
cultivar: Drakkar
age: 10 weeks
tissue: root
developmental stage: flowering
cultivation type: hydroponically
treatment: full nutrition
|
| Growth protocol |
For hydroponic growth, Brassica napus seeds were germinated on wet filter paper for 1 week. Germ buds were transferred to plastic boxes containing nutrient medium for 10 weeks. Nutrient medium: 0.6 mM NH4NO3, 1 mM Ca(NO3)2*4H2O, 0.04 mM Fe-EDTA, 0.5 mM K2HPO4, 0.5 mM K2SO4, 0.4 mM Mg(NO3)2*6H2O. Micro nutrients added: 0.8 µM ZnSO4*7H2O, 9 µM MnCl2*4H2O, 0.1 µM Na2MoO4*2H2O, 23 µM H3BO3, 0.3 µM CuSO4*5H2O. The pH was adjusted to 4.7 with 37 % HCl. Nutrient solutions were changed after 4 weeks, and then renewed once a week. After 5 to 6 weeks, media were constantly aerated by an aquarium air pump.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 100 mg frozen material of root tissue was isolated by using Trizol reagent (Invitrogen) according to manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
Microarray assay was performed using the service provider LC Sciences (Houston, TX). The assay started from 2 to 5 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (from Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 µL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
|
| Scan protocol |
Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
Chip contained 653 plant microRNA probes (Sanger miRBase 10.1) plus additional 85 custom probes for the detection of small RNA sequences derived from Brassica napus phloem sap (Bn_PsRNAs).
|
| Data processing |
Data were analyzed by first subtracting the background and then normalizing the signals by removing system related variation (sample amount variations, different labeling dyes and signal gain differences of scanners) using a locally-weighted regression filter (cyclic LOWESS). For two color experiments, the ratio of the two sets of detected signals (log2 transformed, balanced) and p-values of the t-test were calculated; differentially detected signals were those with less than 0.01 p-values.
The VALUE column represents the statistically significant log2 ratios of Cy3 (-Fe) and Cy5 (FN) labeled transcripts [Log2 (Signal Cy3 / Signal Cy5)].
|
| |
|
| Submission date |
Feb 10, 2010 |
| Last update date |
Feb 23, 2010 |
| Contact name |
Anja Buhtz |
| E-mail(s) |
anja.buhtz@upm.es
|
| Organization name |
Centro de Biotecnologia y Genomica de Plantas
|
| Lab |
280
|
| Street address |
Campus de Montegancedo, M40,km38
|
| City |
Madrid |
| ZIP/Postal code |
28223 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9737 |
| Series (1) |
| GSE20263 |
Small RNAs in different tissues and phloem of oilseed rape under nutrient stress conditions |
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