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Sample GSM507528 Query DataSets for GSM507528
Status Public on Feb 24, 2010
Title Brassica napus_leaf_Cu-starvation (2 weeks) vs. full nutrition
Sample type RNA
 
Channel 1
Source name Brassica napus, leaf, 2 weeks without copper
Organism Brassica napus
Characteristics cultivar: Drakkar
age: 10 weeks
tissue: leaf
developmental stage: flowering
cultivation type: hydroponically
treatment: copper starvation
Treatment protocol Copper starvation was applied for two weeks before flowering started by changing to medium without copper. Here, the 0.3 µM CuSO4*5H2O were omitted from the full nutrient solution.
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 leaf 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, leaf, full nutrition
Organism Brassica napus
Characteristics cultivar: Drakkar
age: 10 weeks
tissue: leaf
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 leaf 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 714 plant microRNA probes (Sanger miRBase 11.0) 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 (-Cu) 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 GPL9738
Series (1)
GSE20263 Small RNAs in different tissues and phloem of oilseed rape under nutrient stress conditions

Data table header descriptions
ID_REF
Signal Leaf -Cu Signal value (background-substracted and normalized) under Cu starvation (-Cu)
StDev Leaf -Cu Standard deviation
Signal Leaf FN Signal value (background-substracted and normalized) under full nutrition (FN)
StDev Leaf FN Standard deviation
p-value statistic parameter that measures the similarity of Cy3 and Cy5 labeled transcripts
VALUE statistically significant log2 ratio of Cy3 (-Cu) and Cy5 (FN) labeled transcripts, Log2 (Signal Leaf –Cu / Signal Leaf FN)

Data table
ID_REF Signal Leaf -Cu StDev Leaf -Cu Signal Leaf FN StDev Leaf FN p-value VALUE
1 17.42 2.27 12.88 9.28 0.782
2 11.28 3.18 5.28 6.48 0.649
3 12.75 3.59 4.69 8.98 0.607
4 1 1.49 14.13 14.82 0.458
5 1 1.1 14.92 12.58 0.38
6 13.84 3.89 5.92 14.83 0.723
7 10.56 4.03 3.48 6.28 0.595
8 5.04 2.42 2.52 8.37 0.881
9 1 1.48 1 19.11 0.998
10 1.17 1.4 1.47 12.82 0.96
11 61.06 11 55.92 14.31 0.85
12 41.83 10.35 49.88 15.64 0.71
13 45.41 5.23 48.55 14.2 0.85
14 23.02 5.17 45.33 10.36 0.169
15 32.53 6.46 49.71 17.3 0.445
16 111.28 13.43 55.94 11.74 0.0171
17 65.13 9.19 40.77 14.19 0.282
18 76.89 10.32 43.74 16.69 0.19
19 56.94 5.84 41.08 12.75 0.425
20 69.37 11.55 48.82 15.05 0.411

Total number of rows: 3281

Table truncated, full table size 107 Kbytes.




Supplementary file Size Download File type/resource
GSM507528.txt.gz 69.6 Kb (ftp)(http) TXT
Processed data included within Sample table

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