4 day old wild-type seedlings, 8h DEX+CHX treatment
Treatment protocol
Dexamethasone (DEX) and cycloheximide (CHX; Sigma-Aldrich, Zwijndrecht, The Netherlands) were dissolved in 70% (v/v) ethanol as 10 mM stocks and used at a final concentration of 10 µM for both liquid and solid media. Wild-type and 35S:BBM GR seeds were germinated on a 150 micron nylon mesh placed on top of 0.5X MS-10 agar plates. Four days later, the nylon mesh with the germinated seedlings was transferred to a 9 cm Petri dish containing 5 ml water supplemented with either 10 µM DEX and 10 µM CHX or the corresponding amount of 70% ethanol. Seedlings were incubated in either solution with gentle rotation for 8 hours, and then frozen in liquid nitrogen and stored at 80C. To ensure the DEX responsiveness of the 35S:BBM GR transgenic lines, the same seed batch was germinated directly on solid 0.5X MS-10 medium containing 10 µM DEX and then scored for somatic embryo formation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Qiagen RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RNA quality was monitored by agarose gel electrophoresis and spectrophotometry. 35S:BBM GR lines were grown in duplicate and RNA was isolated for each sample. The RNA from wild-type seedlings treated with DEX+CHX was isolated from 4 different plates and pooled after assessing RNA quality.
Label
Cy3
Label protocol
For target labeling, 1 ?g of seedling total RNA was linearly amplified in the presence of 5-(3-aminoallyl)-UTP (Sigma-Aldrich) using the MessageAmp aRNA Kit (Ambion Ltd., Huntingdon, UK). Cy3 and Cy5 mono-Reactive Dyes (Amersham Biosciences, Rosendaal, The Netherlands) were coupled to the amplified RNA (aRNA) in freshly made 0.1M sodium carbonate buffer, pH 9.3 for 30 min at room temperature. Labeled aRNA was fragmented prior to hybridization using RNA Fragmentation Reagents (Ambion). RNA amplification, labeling and fragmentation efficiencies were monitored by agarose gel electrophoresis and by measurement of the Cy5 and Cy3 fluorescence emissions at 635 and 532 nm, respectively, using a Molecular Imager FX Pro Plus scanner (BioRad Laboratories Inc. Veenendaal, The Netherlands).
4 day old wild-type seedlings (sample B), 8h mock treatment
Treatment protocol
Dexamethasone (DEX) and cycloheximide (CHX; Sigma-Aldrich, Zwijndrecht, The Netherlands) were dissolved in 70% (v/v) ethanol as 10 mM stocks and used at a final concentration of 10 ?M for both liquid and solid media. Wild-type and 35S:BBM GR seeds were germinated on a 150 micron nylon mesh placed on top of 0.5X MS-10 agar plates. Four days later, the nylon mesh with the germinated seedlings was transferred to a 9 cm Petri dish containing 5 ml water supplemented with either 10 ?M DEX and 10 ?M CHX or the corresponding amount of 70% ethanol. Seedlings were incubated in either solution with gentle rotation for 8 hours, and then frozen in liquid nitrogen and stored at 80C. To ensure the DEX responsiveness of the 35S:BBM GR transgenic lines, the same seed batch was germinated directly on solid 0.5X MS-10 medium containing 10 ?M DEX and then scored for somatic embryo formation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Qiagen RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RNA quality was monitored by agarose gel electrophoresis and spectrophotometry. 35S:BBM GR lines were grown in duplicate and RNA was isolated for each sample. The RNA from wild-type seedlings treated with DEX+CHX was isolated from 4 different plates and pooled after assessing RNA quality.
Label
Cy5
Label protocol
For target labeling, 1 ?g of seedling total RNA was linearly amplified in the presence of 5-(3-aminoallyl)-UTP (Sigma-Aldrich) using the MessageAmp aRNA Kit (Ambion Ltd., Huntingdon, UK). Cy3 and Cy5 mono-Reactive Dyes (Amersham Biosciences, Rosendaal, The Netherlands) were coupled to the amplified RNA (aRNA) in freshly made 0.1M sodium carbonate buffer, pH 9.3 for 30 min at room temperature. Labeled aRNA was fragmented prior to hybridization using RNA Fragmentation Reagents (Ambion). RNA amplification, labeling and fragmentation efficiencies were monitored by agarose gel electrophoresis and by measurement of the Cy5 and Cy3 fluorescence emissions at 635 and 532 nm, respectively, using a Molecular Imager FX Pro Plus scanner (BioRad Laboratories Inc. Veenendaal, The Netherlands).
Hybridization protocol
Labeled target samples were hybridized to 26,000 element Arabidopsis Oligonucleotide Microarrays (Qiagen-Operon Arabidopsis Genome Oligo Set Version 1.0,Qiagen Operon, Alameda, CA, USA), which were printed and provided by the University of Arizona. Immobilization of the oligonucleotide array elements was performed as described at http://ag.arizona.edu/microarray/protocol1.doc. Slides were pre-hybridized at 50C in 120 ?l SlideHyb Glass Array Hybridization Buffer #1 (Ambion) using the HybArray hybridization station (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA). The pre-hybridization mixture was replaced after 2 hours by 120 ?l pre-warmed SlideHyb Glass Array Hybridization Buffer #1 (Ambion) containing heat-denatured labeled target (2.5 ?g Cy3-labeled aRNA and 1.25 ?g Cy5-labeled aRNA) and incubated overnight at 50C. The slides were then washed at room temperature down to 0.1x SSC in the hybridization station.
Scan protocol
Slides were scanned separately for the two fluorescent dyes with a GenePix 4000B scanner (Axon Instruments, Union City, CA, USA). The integrated optical density of each probe was measured using the AIS software (Imaging Research Inc., St Catharines, Ontario, Canada).
Description
Hybridizations were performed using four slides with Cy3-labeled wild-type RNA (WT DC) versus Cy5-labeled 35S:BBM GR RNA from the replicated samples of each of the two transgenic lines (GR#1A DC, GR #1B DC, GR #2A DC and GR #2B DC). The target labeling was reversed for one technical replicate from each transgenic line on two additional slides (GR#1A DC and GR #2A DC versus WT DC). Cy3-labeled WT DC RNA was hybridized in duplicate with Cy5-labeled wild-type RNA from untreated biological replicates (WTA and WTB) on two slides in a control experiment designed to rule out nonspecific induction due to DEX+CHX treatment as opposed to BBM activation. An additional control hybridization was performed in duplicate with RNA from untreated samples of both GR lines (GR#1 and GR#2) labeled with Cy3 and wild-type biological replicates pooled before labeling with Cy5 to identify background expression solely due to the 35S:BBM-GR transgene.
Data processing
Normalization was performed using the median of the background corrected ratios for all spots excluding negative controls and blanks. Differential expression in each hybridization experiment, expressed as the 2Log value of the normalized signal ratios, was tested for statistical significance using a simple t-test per clone, an Analysis Of Variance (Anova) and SAM (Significance Analysis of Microarrays; Tusher et al., 2000).
