Weevils were kept without food on moist filter paper for 48 hr before they were placed on the trees. Five weevils were placed on each tree and caged on the lower stem section corresponding to the first year of growth. Bark tissue from the area of weevil feeding was harvested 6 hours after treatment.
Growth protocol
Three year old seedlings were grown outside of the University of British Columbia (UBC) green house and transferred to the UBC greenhouse one week prior to treatments. Standard greenhouse conditions are described in Ralph et al (2006) Plant Cell & Environment 29: 1545-1570.
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 by spectrophotometer and the quality was evaluated by 2100 Bioanalyzer (Agilent Technologies). RNA was also evaluated for the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer. The resulting cDNA smear was evaluated using gel electrophoresis.
Label
Cy3
Label protocol
Hybridizations were performed using the Genisphere Array900 kit (Genisphere, Hatfield, USA) following manufacturer's instructions. Ten micrograms 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 65ºC for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).
Untreated control collected at 6 hours of the experiment time course.
Growth protocol
Three year old seedlings were grown outside of the University of British Columbia (UBC) green house and transferred to the UBC greenhouse one week prior to treatments. Standard greenhouse conditions are described in Ralph et al (2006) Plant Cell & Environment 29: 1545-1570.
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 by spectrophotometer and the quality was evaluated by 2100 Bioanalyzer (Agilent Technologies). RNA was also evaluated for the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer. The resulting cDNA smear was evaluated using gel electrophoresis.
Label
Cy5
Label protocol
Hybridizations were performed using the Genisphere Array900 kit (Genisphere, Hatfield, USA) following manufacturer's instructions. Ten micrograms 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 65ºC 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 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt's solution, 1 mM EDTA, 2.75 μL LNA d(T) blocker, 2 μg sheared salmon testes DNA (Invitrogen) and 0.3 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 2x 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 95ºC in MilliQ-H2O, and dried by centrifugation (3 min 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 80ºC for 10 min, then maintained at 65ºC prior to adding to a microarray slide heated to 55ºC, covered with a 22 x 60 x 1.5 mm glass coverslip (Fisher Scientific), and incubated for 16 h at 60ºC. Arrays were washed in 2x SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65ºC in the same solution, then three washes of 5 min in 2x SSC at room temperature, and three washes of 5 min in 0.2x 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 0.25M NaPO4, 0.5% SDS, 1x SSC, 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 60ºC, 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).
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity. After quantification of the signal intensities, data were normalized using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18:S96-S104. Four hybridizations were performed for treatment and control comparison within each time point (6 hours, 2 days, 2 weeks) and one hybridization was performed for each comparison between time points for both treatment and control. The experiment was designed to be Cy3 Cy5 dye balanced. To calculate changes in gene expression a linear mixed-effect model was used for 18 microarray slides (hybridizations) of the experiment. P values were calculated for each EST for treatment vs. control comparison and q values were calculated to correct for false discovery rate for each p value (Storey and Tibshirani, 2003). All statistical analyses were done using the R statistical package (www.r-project.org/).
