For the experiment, plantlets were grown in vitro in stacked doubled magenta vessel (Sigma, Oakville, ON, Canada). Each vessel contained a single plantlet. Axillary apex of single node cuttings were grown for 20 days on MS medium containing 8% bacto-agar (BD Diagnostic System, Sparks, MD, USA), and 3% sucrose. In the growth room, plantlets were submitted to a 16-h photoperiod at 60 μmol photons m-2 s-1 and 23/20°C day/night temperature. After 20 days, the plantlets second leaf was harvested.
Extracted molecule
total RNA
Extraction protocol
The second leaves from tomato plantlets grown under in vitro culture conditions were harvested after 20 days in vitro. Tissue were immediately frozen in liquid nitrogen and stored at -80˚C for further grinding. Total RNA was isolated using Plant RNA Reagent following manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). RNA concentration was measured using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). Possible contamination was access with the 260/280nm ratio. RNA quality was visualized with a 1.5% denatured agarose gels.
Label
Cy3
Label protocol
cDNA was produced by reverse transcription of 40 μg of total RNA using the Superscript III (Invitrogen). Aminoallyl-dUTP (Applied Biosystems, Streetsville, ON, Canada) were added to the cDNA synthesis reaction to make possible further indirect labelling. The RNA of the hybrid cDNA-RNA was hydrolysed by addition of NaOH 1M and 15 min heating at 65ºC. Reaction was neutralized by adding HCL 1M. A purification step was realized using the Qiaquick PCR purification kit (Qiagen, Mississauga, ON, Canada). Sample where labelled using AlexaFluor 555 reactive dye packs (Invitrogen) according to the manufacturer. Briefly, 1 μg of cDNA from each sample were evaporated with Savant speedvac concentrator (Thermo Fisher Scientific, Waltham, MA), resuspended in 5 μl double-distilled water and 3 μl labelling buffer. DMSO (2 μl) was added to each single-use fluorophore vial and vortexed. Sample cDNA was added to AlexaFluor 555 reactive dye vial. Labelling was performed for a three hour period. Following a purification step, 600 ng of labelled cDNA of each probes were speedvac-evaporated.
Axillary apex of single node cuttings were grown for 20 days on MS medium containing 8% bacto-agar (BD Diagnostic System, Sparks, MD, USA), and 3% sucrose. In the growth room, plantlets were submitted to a 16-h photoperiod at 60 μmol photons m-2 s-1 and 23/20°C day/night temperature. After 20 days, plantlets were transferred ex vitro for a 12 days acclimatization. For the first 4 days, tomato plants were maintained in an Arabidopsis chamberTM (Conviron, Winnipeg, MB, Canada) under high humidity condition and 16-h photoperiod at 200 μmol photons m-2 s-1. Plantlets were transferred to a greenhouse under natural daylight (august) for the remaining 8 days in order to complete the acclimatization process. Plantlets second leaf was harvested after 12 days ex vitro. The second leaf corresponded to a newly ex vitro formed leaf by opposition to old, permanent in vitro leaves
Extracted molecule
total RNA
Extraction protocol
After 12 days of growth ex vitro, the second leaves of tomato plant were harvested. Tissue were immediately frozen in liquid nitrogen and stored at -80˚C for further grinding. Total RNA was isolated using Plant RNA Reagent following manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). RNA concentration was measured using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). Possible contamination was access with the 260/280nm ratio. RNA quality was visualized with a 1.5% denatured agarose gels.
Label
Cy5
Label protocol
cDNA was produced by reverse transcription of 40 μg of total RNA using the Superscript III (Invitrogen). Aminoallyl-dUTP (Applied Biosystems, Streetsville, ON, Canada) were added to the cDNA synthesis reaction to make possible further indirect labelling. The RNA of the hybrid cDNA-RNA was hydrolysed by addition of NaOH 1M and 15 min heating at 65ºC. Reaction was neutralized by adding HCL 1M. A purification step was realized using the Qiaquick PCR purification kit (Qiagen, Mississauga, ON, Canada). Sample where labelled using AlexaFluor 647 reactive dye packs (Invitrogen) according to the manufacturer. Briefly, 1 μg of cDNA from each sample were evaporated with Savant speedvac concentrator (Thermo Fisher Scientific, Waltham, MA), resuspended in 5 μl double-distilled water and 3 μl labelling buffer. DMSO (2 μl) was added to each single-use fluorophore vial and vortexed. Sample cDNA was added to AlexaFluor 647 reactive dye vial. Labelling was performed for a three hour period. Following a purification step, 600 ng of labelled cDNA of each probes were speedvac-evaporated.
Hybridization protocol
SlideHyb buffer #1 (Applied Biosystems) was preheated to 68ºC for 30 minutes. Labelled cDNA was resuspended in 4 μl 10 mM EDTA and heat denatured at 95ºC for 10 min. SlideHyb buffer #1 was added to the labelled cDNA. Hybridization was performed by using a SlideBooster from Advalytix AG (Brunnthal, Germany). For this purpose, a lifterSlip (Erie Scientific Company, Partsmouth, NH) was laid down on the array and the hybridization solution injected. After 17-h of hybridization at 55ºC, slides were washed in a low and high stringency buffer as mentioned in the SlideHyb buffer #1 protocol.
Scan protocol
Slides were scanned using a VersArray ChipReader (BioRad, Mississauga, ON, Canada). Digital image were acquired with the ChipReader 3.1 software (Bio-Rad).
Description
Hybridization and study was conducted to identify genes regulated by the in vitro culture conditions.
Data processing
Data image were transferred to the Array-Pro Analyzer v.4.5 software (Media Cybernetics, Silver Spring, MD, USA) to detect the intensity of each spot on the array and for background subtraction using the local ring method. Net intensity data were log2 transformed. Data were normalized using the global Loess method included in the Microarray Analysis of Variance (MAANOVA) software. Normalization was performed in order to consider possible intensity-dependent variations in dye bias. Statistical analysis was accomplished by using the F-test of the R/maanova package (Wu et al., 2002), which work under the R programming environment. The false discovery rate (FDR) procedure was applied to the p-values in order to minimize the number of false positives associated to multiple testing. Differentially expressed genes were selected according to the adjusted p-values. Only genes showing an adjusted p-value under 0.01 were kept for further analysis. In addition, the fold change in expression between the two treatments had to be higher than 1.5 to be considered.
