Investigation Title	Transcription profiling of ethylene-dependent responses to short day exposure in birch apices	
Comment[Submitted Name]	Ethylene-dependent responses to short day exposure in birch apices	
Experimental Design	growth_condition_design	transcription profiling by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]	2005-11-23	
Comment[AEMIAMESCORE]	3	
Comment[ArrayExpressAccession]	E-MEXP-491	
Comment[MAGETAB TimeStamp_Version]	2010-08-11 14:36:13 Last Changed Rev: 13058	
Experimental Factor Name	ethylene sensitivity	strain	time	
Experimental Factor Type	phenotype	strain_or_line	time	
Experimental Factor Term Source REF	
Person Last Name	Ruonala	
Person First Name	Raili	
Person Mid Initials	
Person Email	raili.ruonala@helsinki.fi	
Person Phone	+358 9 19159439	
Person Fax	
Person Address	POB 65 (Viikinkaari 1)	
Person Affiliation	Department of Biological and Environmental Science	
Person Roles	submitter	
Person Roles Term Source REF		
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2005-11-23	
PubMed ID	16640599	
Publication DOI	16640599	
Publication Author List	
Publication Title	
Publication Status	journal_article	
Publication Status Term Source REF	
Experiment Description	We have used transgenic ethylene-insensitive birches (Betula pendula), which express the Arabidopsis ethylene receptor gene ETR1 carrying the dominant mutation etr1-1, to investigate the role of ethylene in short day (SD) -induced responses in the shoot apical meristem in birch. Wild-type birch (clone V5834) and two ethylene-insensitive lines in this background (BPetr1-1-35 and BPetr1-1-86; see Plant Physiol 132: 185-195) were exposed to SD. After 12, 16 and 20 days under SD, apices of branches of three trees were pooled before RNA extraction from each sample. To study the ethylene-dependent SD-transcriptome in birch apices, the RNA extracts of lines BPetr1-1-35 and BPetr1-1-86 were separately compared with the reference, wild-type V5834, at the three time points (12SD, 16SD, 20SD) resulting in altogether six microarray hybridizations.	
Protocol Name	P-MEXP-15020	P-MEXP-15023	P-MEXP-15021	P-MEXP-15022	P-MEXP-15024	P-MEXP-15025	P-MEXP-15026	P-MEXP-15027	P-MEXP-15047	P-MEXP-15048	
Protocol Type	grow	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	pool	nucleic_acid_extraction	labeling	labeling	hybridization	feature_extraction	
Protocol Description	Birch (Betula pendula) shoots of wild-type clone V5834 and two transgenic ethylene-insensitive lines (BPetr1-1-35 and BPetr1-1-86; see Vahala, J., Ruonala, R., Keinänen, M., Tuominen, H., and Kangasjärvi, J.  2003. Ethylene insensitivity modulates ozone-induced cell death in birch. Plant Physiol 132: 185-195) in this clonal background were in vitro -propagated on agar-solidified woody plant medium (Lloyd, G.B., and McCown, B.H. 1980. Commercially feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot-tip culture. Proc Int Plant Proc Soc 30: 421-437) supplied with 1 mg/ml zeatin, and rooted on half-strength Murashige and Skoog -medium (Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473-497) supplied with 0.5 mg/ml indolyacetic acid. Copies of the clones were transplanted into 1l-pots in peat:sand:vermiculite (6:2:1, v/v/v), fertilized with 2.5 g/l of Osmocote Exact Hi-Start (Scotts, Heerlen, The Netherlands; N:P:K 15:4:8) and grown for seven weeks in greenhouse prior to experiments. In the greenhouse, natural light was supplemented with high pressure sodium lamps (Lucalox LU400/HO/T/40, GE Lighting, Hungary) to give a minimum of 200 µmol m-2 s-1 photosynthetic photon flux and a day length of 18 hours, with a temperature of 18°C and a relative humidity of 70%. All conditions in the greenhouse were regulated and monitored by a computer controlled system (Priva Intégro, Canadian Climatrol Systems Ltd, Canada). Growing conditions were identical for all plants, and the potential effects of microclimate inside the greenhouse compartment were avoided by regularly varying the position of the plants.	For the short day (SD) exposure, seven-week-old, greenhouse-grown birches (wild-type V5834 and ethylene-insensitive lines BPetr1-1-35 and BPetr1-1-86) were transferred to a greenhouse compartment where day length was shortened to 12 hours by using curtains regulated and monitored by a computer controlled system (Priva Intégro, Canadian Climatrol Systems Ltd, Canada). After 20 days under SD, sampling was performed. Plant material was sampled during the middle of the photoperiod. All visible leaves surrounding the apex were dissected with a scalpel and the stem was cut approximately three mm below the shoot apex. The samples were frozen in liquid nitrogen and stored at -80°C until RNA isolation.	For the short day (SD) exposure, seven-week-old, greenhouse-grown birches (wild-type V5834 and ethylene-insensitive lines BPetr1-1-35 and BPetr1-1-86) were transferred to a greenhouse compartment where day length was shortened to 12 hours by using curtains regulated and monitored by a computer controlled system (Priva Intégro, Canadian Climatrol Systems Ltd, Canada). After 12 days under SD, sampling was performed. Plant material was sampled during the middle of the photoperiod. All visible leaves surrounding the apex were dissected with a scalpel and the stem was cut approximately three mm below the shoot apex. The samples were frozen in liquid nitrogen and stored at -80°C until RNA isolation.	For the short day (SD) exposure, seven-week-old, greenhouse-grown birches (wild-type V5834 and ethylene-insensitive lines BPetr1-1-35 and BPetr1-1-86) were transferred to a greenhouse compartment where day length was shortened to 12 hours by using curtains regulated and monitored by a computer controlled system (Priva Intégro, Canadian Climatrol Systems Ltd, Canada). After 16 days under SD, sampling was performed. Plant material was sampled during the middle of the photoperiod. All visible leaves surrounding the apex were dissected with a scalpel and the stem was cut approximately three mm below the shoot apex. The samples were frozen in liquid nitrogen and stored at -80°C until RNA isolation.	Of each tree, the apices of axillary branches were collected. For each sample, apices of branches were pooled from three trees before RNA extraction.	RNA was isolated from Betula pendula apices using the method described by Chang et al., 1993. (Chang, S., Puryear, J., and Cairney, J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11: 113-116.)	15 mg total-RNA was mixed with 1 ml amino-oligodT (2 mg/ml) in a final volume of 16.5 ml, heated for 10 min at 65°C and cooled down on ice for 1 min. A mastermix for reverse transcription was prepared (per sample); 6 ml 5×first strand buffer; 3 ml DTT; 3 ml 10×aminoallyl dNTP mix. (5mM dATP, dCTP, dGTP, 3mM dTTP, 2 mM aminoallyl-dUTP); 0.5 ml Super RNAse Inhibitor (Ambion) and 1 ml SuperScript III (Invitrogen) per reaction. 13.5 ml mastermix was added to every sample. The reactions were incubated at 46°C for 1 hour. 1 ml MMLV-RT RNAse H- point mutant (Promega) per reaction was added and the incubation continued for 1 hour at 42°C. The RNA was degraded by addition of 2 ml fresh 5M NaOH and 8 ml 0.5M EDTA and incubation for 15 minutes at 65°C. To neutralize the reaction 20 ml 1M HEPES pH 7.2 was added and the cDNA purified on Qiagen PCR columns (Qiagen, Hilden, Germany) and eluted in water. The volume was adjusted in a speed-vac to 27 ml and 3 ml 1M NaHCO3 pH 9.3 was added. A tube of dye ester (Cy3), Amersham Biosciences, Little Chalfont, UK) was dissolved in 73 ml DMSO; and 4.5 ml Cy3-dye was added to the cDNA. The coupling reaction was incubated 2 hours in room temperature with slow shaking (in darkness). Unbound dye was quenched by addition of 4.5 ml 4M hydroxylamine to each reaction and incubation for 15 minutes in the dark at room temperature. Cy3 and Cy5 reactions were combined and purified on Qiagen columns and dried down in a speed-vac.	15 mg total-RNA was mixed with 1 ml amino-oligodT (2 mg/ml) in a final volume of 16.5 ml, heated for 10 min at 65°C and cooled down on ice for 1 min. A mastermix for reverse transcription was prepared (per sample); 6 ml 5×first strand buffer; 3 ml DTT; 3 ml 10×aminoallyl dNTP mix. (5mM dATP, dCTP, dGTP, 3mM dTTP, 2 mM aminoallyl-dUTP); 0.5 ml Super RNAse Inhibitor (Ambion) and 1 ml SuperScript III (Invitrogen) per reaction. 13.5 ml mastermix was added to every sample. The reactions were incubated at 46°C for 1 hour. 1 ml MMLV-RT RNAse H- point mutant (Promega) per reaction was added and the incubation continued for 1 hour at 42°C. The RNA was degraded by addition of 2 ml fresh 5M NaOH and 8 ml 0.5M EDTA and incubation for 15 minutes at 65°C. To neutralize the reaction 20 ml 1M HEPES pH 7.2 was added and the cDNA purified on Qiagen PCR columns (Qiagen, Hilden, Germany) and eluted in water. The volume was adjusted in a speed-vac to 27 ml and 3 ml 1M NaHCO3 pH 9.3 was added. A tube of dye ester (Cy5), Amersham Biosciences, Little Chalfont, UK) was dissolved in 73 ml DMSO; and 4.5 ml Cy5-dye was added to the cDNA. The coupling reaction was incubated 2 hours in room temperature with slow shaking (in darkness). Unbound dye was quenched by addition of 4.5 ml 4M hydroxylamine to each reaction and incubation for 15 minutes in the dark at room temperature. Cy3 and Cy5 reactions were combined and purified on Qiagen columns and dried down in a speed-vac.	The arrays are crosslinked at 90 mJ/cm2. Prepare prehybridisation buffer (50 ml 20*SSC, 2g BSA, 150 ml water, 1 ml 20% SDS) and preheat 30 min 42ºC. Pre-hybridise slides for 30min-2h at 42ºC. Wash slides 30 seconds in MilliQ-water and 30 seconds in 95% EtOH. Spin the arrays dry for 5 min, 500 rpm (Eppendorf 5810R). Immediately put array in hybridization chamber and apply washed LifterSlip and apply probe. The LifterSlip (24*60mm, Erie Scientific) is washed in 1%SDS, water, 95% EtOH and airdried. Resuspend probe in 25 ul formamide, 12.5 ul 20*SSC, 2.5 ul 2% SDS, 5 ul herring sperm DNA (1 mg/ml) and 5 ul 50* Denhardts. Final volume should be 50ul. Heat 2 min 96ºC, spin 30s full speed. Apply the probe to the array. Hybridize at 42ºC overnight (14-16 hours). After hybridization the microarrays were briefly dipped in 2xSSC/0.1%SDS until the LifterSlip falls off. The arrays were washed in 2×SSC, 0.1% SDS for 3 min, 1× SSC for 2 min, 0.2× SSC for 1 min, 0.05×SSC for 10 seconds . Finally, the washed slides were dried by centrifuging for 5 min, 500 rpm (Eppendorf 5810R). All washing steps were performed at room temperature.	A prewarmed (15 min) ScanArray 5000 were used for scanning. First a prescan of the first blocks was performed with initial settings of 90% laser power and 80% PMT gain. Then a line scan was performed and the laser power settings were adjusted to get as equal Cy3 and Cy5 channel intensity as possible. Finally the real scan was done with 5 um resolution.	
Protocol Parameters						Amplification;Extracted product;	Label used;Amount of nucleic acid labeled;Amplification;	Amplification;Label used;Amount of nucleic acid labeled;	temperature;Quantity of label target used;Chamber type;	
Protocol Hardware										ScanArray 5000 [PerkinElmer]	
Protocol Software										ScanArray Express [PerkinElmer]	
Protocol Contact	
Protocol Term Source REF		mo	mo	mo	
SDRF File	E-MEXP-491.sdrf.txt	
Term Source Name	po		ArrayExpress	mo	EFO	
Term Source File	http://www.plantontology.org		http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version