genotype: CCLONE 45226 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 20g of DNase treated total RNA annealed to 2L anchored oligo(dT)20 primer plus 1L 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 500L wash buffer. cDNA was eluted twice with 50L DEPC-water, EtOH precipitated ( 10L of 3M Sodium Acetate, pH5.2, 2L 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 5L 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 80L 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 65L 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.
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 20g of DNase treated total RNA annealed to 2L anchored oligo(dT)20 primer plus 1L 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 500L wash buffer. cDNA was eluted twice with 50L DEPC-water, EtOH precipitated ( 10L of 3M Sodium Acetate, pH5.2, 2L 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 5L 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 80L 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 65L 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 20L 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
45226_WW_4B
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.