tissue: sink leaf genotype: H11-11 experimental protocol: Six month old clonal trees grown under greenhouse conditions. Leaves at the crown with a midvein length < 6cm were harvested with petioles removed from five trees subjected to the same treatment. Leaves act as carbon sinks in relation to systemic source and treated source leaves. RNA was isolated individually from each of the five trees and then pooled prior to cDNA synthesis.
Biomaterial provider
Shoots were harvested from trees growing at the University of British Columbia South Campus farm.
Treatment protocol
Five lowest, fully-expanded, healthy source leaves were caged on each tree using nylon mesh bags. For oral secretion treatments, four 10 cm strips were punctured parallel to the midrib on each of the five caged leaves with a pattern wheel, and 20 microL of forest tent caterpillar oral secretion was also applied to the wound sites. Systemic sink leaves were harvested 24 hours after the initiation of treatment.
Growth protocol
Trees were grown under standard greenhouse conditions. See Ralph et al. (2006) Molecular Ecology 15: 1275-1297.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated according to procedures described in Kolosova et al. (2004) (Biotechniques 36: 821-824). Total RNA was quantified and quality checked by spectrophotometer and agarose gel. Poly(A)+ RNA was isolated by oligo d(T) cellulose using the Poly(A) Pure kit (Ambion, Austin, USA), following manufacturer's instructions. RNA was also evaluated for integrity and the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer and _P32 dGTP incorporation. After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns, Amersham Pharmacia Biotech, Buckinghamshire, UK) the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech).
Label
Cy5
Label protocol
Hybridizations were performed using the Genisphere Array350 kit (Genisphere) following manufacturer’s instructions. Forty µg total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5’ unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65°C for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).
tissue: sink leaf genotype: H11-11 experimental protocol: Six month old clonal trees grown under greenhouse conditions. Leaves at the crown with a midvein length < 6cm were harvested with petioles removed from five trees subjected to the same treatment. Leaves act as carbon sinks in relation to systemic source and treated source leaves. RNA was isolated individually from each of the five trees and then pooled prior to cDNA synthesis.
Biomaterial provider
Shoots were harvested from trees growing at the University of British Columbia South Campus farm.
Treatment protocol
Five lowest, fully-expanded, healthy source leaves were caged on each tree using nylon mesh bags. For oral secretion treatments, four 10 cm strips were punctured parallel to the midrib on each of the five caged leaves with a pattern wheel, and 20 microL of forest tent caterpillar oral secretion was also applied to the wound sites. Systemic sink leaves were harvested 2 hours after the initiation of treatment.
Growth protocol
Trees were grown under standard greenhouse conditions. See Ralph et al. (2006) Molecular Ecology 15: 1275-1297.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated according to procedures described in Kolosova et al. (2004) (Biotechniques 36: 821-824). Total RNA was quantified and quality checked by spectrophotometer and agarose gel. Poly(A)+ RNA was isolated by oligo d(T) cellulose using the Poly(A) Pure kit (Ambion, Austin, USA), following manufacturer's instructions. RNA was also evaluated for integrity and the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer and _P32 dGTP incorporation. After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns, Amersham Pharmacia Biotech, Buckinghamshire, UK) the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech).
Label
Cy3
Label protocol
Hybridizations were performed using the Genisphere Array350 kit (Genisphere) following manufacturer’s instructions. Forty µg total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5’ unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65°C for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).
Hybridization protocol
Following pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45 µL hybridization solution consisting of 25% formamide, 4x SSC, 0.5% SDS, 2x Denhardt’s solution, 1 mM EDTA, 4.0 µL LNA d(T) blocker, 2 µg sheared salmon testes DNA (Invitrogen) and 0.75 µL of Cy5-labeled GFP cDNA (Cy5-dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech). Immediately prior to use, arrays were pre-washed 3´ in 0.1% SDS at room temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95°C in MilliQ-H2O, and dried by centrifugation (3 minutes at 2000 rpm in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80°C for 10 min, then maintained at 65°C prior to adding to a microarray slide heated to 55°C, covered with a 22 ´ 60 ´ 1.5 mm glass coverslip (Fisher Scientific), and incubated for 16 h at 48°C. Arrays were washed in 2´ SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65°C in the same solution, then three washes of 5 min in 2´ SSC at room temperature, and three washes of 5 min in 0.2´ SSC at room temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in a 45 µL volume consisting of 25% formamide, 4x SSC, 0.5% SDS, 2x Denhardt’s solution, 1 mM EDTA,, 2.5 µL Cy3 capture reagent and 2.5 µL Cy5 capture reagent. The 3DNA capture reagent is bound to its complementary cDNA capture sequence on the Cy3 or Cy5 oligo d(T) primers. The second hybridization was performed for 3 h at 48°C, then washed and dried as before.
Scan protocol
Fluorescent images of hybridized arrays were acquired by using ScanArray Express (PerkinElmer, Foster City, USA). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively. All scans were performed at the same laser power (90%), but with the photomultiplier tube settings for the two channels adjusted such that the ratio of the mean signal intensities was ~1, and the percentage of saturated array elements was < 0.5% but > 0%, while minimizing background fluorescence. Fluorescent intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El Segundo, USA).
Description
Gene expression.
Data processing
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct for background intensity. After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the vsn method (Huber et al., 2002 Bioinformatics 18: S96-S104). For the oral secretion treatment using pooled biological replicates, total RNA from treated and untreated control leaves of the same source/sink group was compared using a balanced loop consisting of direct and indirect comparisons across treatments and time points, with dye balance, using a total of 54 hybridizations. To assess the transcriptional response to stress treatments, a linear mixed-effects model was fitted to the normalized intensities in the Cy3 and Cy5 channels of each experiment using 54 microarray slides. The model contained an adjustment for dye effect, an array effect indicating which Cy5/Cy3 pair was on each array, and a treatment effect indicating treatment and time point. Expression variance was obtained from technical variance among slides. Next, the ratio of the treatment minus untreated control parameter estimate to the standard error was used to calculate a t statistic and P value. The Q value for each effect and gene was calculated for each of the models to adjust for the false discovery rate (Storey and Tibshirani et al., 2003). All statistical analyses were performed within the R statistical package (version 2.5.1; http://www.r-project.org/).
