Hydrogen cyanamide (HC): 5% Dormex. Heat shock (HS): Incubation for 1 h in 50oC water. Vitis vinefera buds were pooled from multiple single-node cuttings at six time points (3, 6, 12, 24, 48 and 96 h) after control, HS and HC treatments and frozen.
Extracted molecule
total RNA
Extraction protocol
Total RNA samples were extracted separately from each pool according to Or et al.(2000), and PolyA RNA preparations were purified using Dynabeads Oligo (dT)25 (Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions.PolyA RNA samples were then used as templates for amplification reactions using MessageAmpTM II aRNA kit (Ambion, Austin, TX), following the manufacturer's instructions.
Label
Cy5
Label protocol
Direct labeling of the resultant aRNA by random priming was then used for the production of Cy3 and Cy5 probes.A total of 150 µg of aRNA was incubated with 3 µL dNTP (10 mM), 2 µL Cy3 or Cy5-dUTP (1 mM) and 2 µL random hexamer (0.5 µg/µL) in a total volume of 26 µL at 65oC for 5 min.Following the heating step, 8 µL of 5X first-strand buffer, 4 µL of DTT (0.1 M), 1 µL of RNase inhibitor (Invitrogen, Carlsbad , CA) and 2 µL of Power Script (Invitrogen) were added and the reaction was incubated for 2 h at 42oC.Products were purified using RNeasy MinElute columns (Qiagen, Carlsbad, CA) and kept protected from light at -80oC.
Hydrogen cyanamide (HC): 5% Dormex. Heat shock (HS): Incubation for 1 h in 50oC water. Vitis vinefera buds were pooled from multiple single-node cuttings at six time points (3, 6, 12, 24, 48 and 96 h) after control, HS and HC treatments and frozen.
Extracted molecule
total RNA
Extraction protocol
Total RNA samples were extracted separately from each pool according to Or et al.(2000), and PolyA RNA preparations were purified using Dynabeads Oligo (dT)25 (Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions.PolyA RNA samples were then used as templates for amplification reactions using MessageAmpTM II aRNA kit (Ambion, Austin, TX), following the manufacturer's instructions.
Label
Cy3
Label protocol
Direct labeling of the resultant aRNA by random priming was then used for the production of Cy3 and Cy5 probes.A total of 150 µg of aRNA was incubated with 3 µL dNTP (10 mM), 2 µL Cy3 or Cy5-dUTP (1 mM) and 2 µL random hexamer (0.5 µg/µL) in a total volume of 26 µL at 65oC for 5 min.Following the heating step, 8 µL of 5X first-strand buffer, 4 µL of DTT (0.1 M), 1 µL of RNase inhibitor (Invitrogen, Carlsbad , CA) and 2 µL of Power Script (Invitrogen) were added and the reaction was incubated for 2 h at 42oC.Products were purified using RNeasy MinElute columns (Qiagen, Carlsbad, CA) and kept protected from light at -80oC.
Hybridization protocol
For each microarray hybridization, a pair of Cy3- and Cy5-labeled targets, for comparison according to the hybridization plan, were mixed. Microarray cross-linking, hybridization and washing procedures were carried out as detailed at http://ag.arizona.edu/microarray/Microarraymethod1
Scan protocol
Arrays were scanned with a GenePix Microarray scanner (Molecular Devices, Sunnyvale, CA) and quantified with GenePix Pro 5.0 software to extract the median signal and background intensities for each spot for both channels on the microarray.
Description
Due to the small number of replicates, biological-comparison variance was then corrected by empirical Bayesian method to obtain moderated t-statistics (Smyth, 2004) and multiple comparison correction (Benjamini and Hochberg, 1995) was applied. Statistical test of the difference in expression signal between each treatment and control (adjusted p-value < 0.05) was calculated separately for each clone at each time point analyzed.
Data processing
Differentially expressed genes were selected using the Linear Models for Microarray (LIMMA) package which is part of the Bioconductor project (Gentleman et al., 2004). Although it is a dual-channel platform, we analyzed each channel separately as described in chapter 9 of the LIMMA user's guide and presented at the 55th Session of the International Statistics Institute (Smyth, 2005). First, we weighted bad spots by 0.1 and subtracted the local background measurement. This was followed by a per-tip LOWESS normalization within slides (Yang et al., 2002) and variance stabilization normalization (VSN) (Huber et al., 2002) between slides. Technical replicates were averaged and then we estimated the intra-spot correlation between the two channels of the same RNA sample and as weighting for further fitting. This technique is equivalent to a randomized block-over-dye effect within spots (Smyth, 2005).
