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Sample GSM619339 Query DataSets for GSM619339
Status Public on Aug 04, 2011
Title Well Watered CCLONE 44686 Replicate 3 [DS vs. WW]
Sample type RNA
 
Channel 1
Source name 44686_WW3
Organism Pinus taeda
Characteristics genotype: CCLONE 44686
tissue: 10 mo. old propagule roots, pooled from 3 individuals
treatment: Well-Watered (WW)
pre-dawn water potential at harvest: –0.3 MPa ± 0.1
harvest point: Day 9, control, harvested at same time as DR
Treatment protocol Treatment protocol was as described previously: Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16
Growth protocol Tissues samples were isolated from plants grown as described previously:Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16
Extracted molecule total RNA
Extraction protocol RNA Extraction was carried out as described previously:Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16 and Lorenz WW, Yu Y-S, Dean JFD: An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species Journal of Visualized Experiments 2010, 36
Label Cy3
Label protocol First-Strand cDNA Synthesis and Purification Synthesis of amino-allyl and amino-hexyl modified cDNA and was done with an Invitrogen SuperScriptTM Indirect cDNA Labeling System (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol with a few modifications. Briefly, each cDNA synthesis reaction contained 20g of DNase treated total RNA annealed to 2L anchored oligo(dT)20 primer plus 1L random hexamer primer. Reactions were carried out in a 45oC water bath for 12-14 hr. Samples were purified on S.N.A.P™ columns supplied with the kit and washed three times with 500L wash buffer. cDNA was eluted twice with 50L DEPC-water, EtOH precipitated ( 10L of 3M Sodium Acetate, pH5.2, 2L 20mg/mL glycogen, and 300uL -20oC 100% EtOH) at -80oC for 30 min. Pellets were washed three times with -20oC 70% EtOH followed by resuspending in 5L 2X coupling buffer that had been warmed to 43 oC. Cy Dye Coupling and Assessment of Labeling Efficiency Cy-5 and Cy-3 dyes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), were prepared on the same day as labeling by dissolving each in 80L DMSO. Five microliters of dye was added to the resuspended cDNA sample and incubated in the dark for 1 hour. Next, 20uL of 3M sodium acetate, pH5.2 and 500uL loading buffer was added to the dye-coupled cDNA. The sample was passed over a S.N.A.P. column and washed three times as described above. Labeled cDNA was eluted in 65L DEPC-water. Nucleic acid and Cy dye concentrations of labeled target cDNAs were determined using a NanoDrop ND-1000 and concentrations were converted to pmol to calculate a ratio for efficiency of labeling. Typical ratios were in the range of 20-40 pmol n.t./pmol dye and total dye incorporated typically ranged from 200-400 pmol.
 
Channel 2
Source name Reference sample
Organism Pinus taeda
Characteristics reference sample components: CCLONES 40430, 43680, 41586 first flush candle and needle tissue, 500 mg ea.; CCLONES 41201, 41396, 44686, 45226, drought-stressed, drought recovered, and well-watered roots from each genotype, 250 mg ea.; Genotype 7-56, callus tissue (needle origin), 250 mg.
Treatment protocol Treatment protocol was as described previously: Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16
Growth protocol Tissues samples were isolated from plants grown as described previously:Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16
Extracted molecule total RNA
Extraction protocol RNA Extraction was carried out as described previously:Lorenz WW, Sun F, Liang C, Kolychev D, Wang HM, Zhao X, Cordonnier-Pratt MM, Pratt LH, Dean JFD: Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries. Tree Physiology 2006, 26(1):1-16 and Lorenz WW, Yu Y-S, Dean JFD: An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species Journal of Visualized Experiments 2010, 36
Label Cy5
Label protocol First-Strand cDNA Synthesis and Purification Synthesis of amino-allyl and amino-hexyl modified cDNA and was done with an Invitrogen SuperScriptTM Indirect cDNA Labeling System (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol with a few modifications. Briefly, each cDNA synthesis reaction contained 20g of DNase treated total RNA annealed to 2L anchored oligo(dT)20 primer plus 1L random hexamer primer. Reactions were carried out in a 45oC water bath for 12-14 hr. Samples were purified on S.N.A.P™ columns supplied with the kit and washed three times with 500L wash buffer. cDNA was eluted twice with 50L DEPC-water, EtOH precipitated ( 10L of 3M Sodium Acetate, pH5.2, 2L 20mg/mL glycogen, and 300uL -20oC 100% EtOH) at -80oC for 30 min. Pellets were washed three times with -20oC 70% EtOH followed by resuspending in 5L 2X coupling buffer that had been warmed to 43 oC. Cy Dye Coupling and Assessment of Labeling Efficiency Cy-5 and Cy-3 dyes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), were prepared on the same day as labeling by dissolving each in 80L DMSO. Five microliters of dye was added to the resuspended cDNA sample and incubated in the dark for 1 hour. Next, 20uL of 3M sodium acetate, pH5.2 and 500uL loading buffer was added to the dye-coupled cDNA. The sample was passed over a S.N.A.P. column and washed three times as described above. Labeled cDNA was eluted in 65L DEPC-water. Nucleic acid and Cy dye concentrations of labeled target cDNAs were determined using a NanoDrop ND-1000 and concentrations were converted to pmol to calculate a ratio for efficiency of labeling. Typical ratios were in the range of 20-40 pmol n.t./pmol dye and total dye incorporated typically ranged from 200-400 pmol.
 
 
Hybridization protocol Hybridization was carried out as described previously: Lorenz WW, Yu Y-S, Simões M, Dean JFD: Processing the Loblolly Pine PtGen2 cDNA Microarray. Journal of Visualized Experiments 2009, 25. 50 pmol each of Cy-5 and Cy-3 labeled target was combined in a single tube and dried in a vacuum desiccator at 45oC to a volume of 1-2 L. cDNAs were resuspended in 60uL hybridization buffer (30% formamide, 5X SSC, 5X Denhardt’s, 1% PolyA RNA, 0.1% SDS), vortexed briefly and heated in a 95oC water bath for 5 min.. Targets were loaded at the bottom edge of the LifterSlip™ by way of capillary action. Slides were placed in a HybChamber™ (Genomic Solutions, Ann Arbor, MI ) and 20L of 100mM DTT was added to humidity wells located at each end of the chamber. The chamber was assembled, wrapped in aluminum foil, and incubated in a 48oC shaking water bath set at 50 rpm for 14-16 hr.
Scan protocol The microarrays were scanned using a ProScanArray™ confocal scanner (Perkin Elmer, Waltham, MA). Scans were conducted with the 532 nm and 635 nm lasers and data were collected at a 10 m resolution. Laser power remained constant at 90% and gain settings typically ranged between 55-65%.
Description 44686_WW_3B
Data processing Raw fluorescence data were processed with an image analysis algorithm in ImaGene software, ver. 7.5 (Bio-Discovery Inc., El Segundo, CA, USA) was used to quantify signal and background intensity. Probes that had been duplicated on the array as internal controls were flagged to leave a single representative for each duplicated set and signal means were determined without background correction. An in house Perl script was used to import the raw Imagene signal intensity data, to calculate Wang et al. flags (Wang et al. Bioinformatics 2003,19:1341-1347) and to convert the data into a readily exportable file for downstream BRB array tools statistical analysis. Normalization and Statistical Analysis Data was filtered and normalized using BRB-Array Tools, an R-based integrated package for the visualization and statistical analysis of DNA microarray gene expression data (Simon et al. Cancer Informatics 2007, 3:11-17). Using the reference design experimental module, raw signal intensity data from all replicates, 72 slides total, were log2 transformed and normalized by the Lowess plus print tip method. Spot signals were filtered by employing Wang flagging and gene filters were employed to remove any gene with 50% or more missing or filtered data. Normalized signal intensity ratios were calculated for identification of differentially expressed genes (DEGs) that were determined via paired comparisons among the three treatment groups using the class comparison tool and utilizing a random variance t-test for computation of P-values. Control of the proportion of false discoveries was accomplished the multivariate permutation test with the number of permutations at the default setting of 1000. All paired treatment comparison analyses were performed with a false discovery rate (FDR) of 0.01 and a confidence level of 90%. The list DEGs identified in the WW vs. DS comparison with at least a 1.5-fold difference in expression was used to extract the BRB normalized log mean ratio data identified from all three treatments.
 
Submission date Nov 09, 2010
Last update date Aug 04, 2011
Contact name Adam W. Barb
E-mail(s) abarb@uga.edu
Organization name University of Georgia
Department Biochemistry and Molecular Biology
Lab B302A
Street address 180 E. Green Street
City Athens
State/province GA
ZIP/Postal code 30602
Country USA
 
Platform ID GPL11184
Series (1)
GSE25216 Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.).

Data table header descriptions
ID_REF
VALUE Lowess normalized, filtered log2 ratio (test/reference)

Data table
ID_REF VALUE
1.1.1.1
1.1.1.2 0.291241258
1.1.1.3 0.079368971
1.1.1.4 0.071083397
1.1.1.5 -0.100869581
1.1.1.6 -0.02197114
1.1.1.7 -0.105655909
1.1.1.8 0.133184597
1.1.2.1 -0.291536242
1.1.2.2 -0.596389651
1.1.2.3 -1.074280739
1.1.2.4
1.1.2.5 -0.185347438
1.1.2.6 -0.637761235
1.1.2.7 -0.384057641
1.1.2.8 -0.219571859
1.1.2.9 0.149711281
1.1.2.10 -0.043971933
1.1.2.13 -0.525209904
1.1.2.14 0.222272038

Total number of rows: 25378

Table truncated, full table size 526 Kbytes.




Supplementary data files not provided
Processed data included within Sample table
Processed data are available on Series record

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