Investigation Title Transcription profiling of Capsella bursa-pastoris early and late flowering seedlings with and without vernalization
Comment[Submitted Name] UU Capsella bursa-pastoris Flowering ecotype PL vs SE14 seedlings with and without vernalization
Experimental Design loop_design replicate_design strain_or_line_design growth_condition_design transcription profiling by array
Experimental Design Term Source REF mo mo mo EFO
Comment[ArrayExpressReleaseDate] 2007-07-18
Comment[AEMIAMESCORE] 3
Comment[ArrayExpressAccession] E-ATMX-22
Comment[MAGETAB TimeStamp_Version] 2010-10-15 22:30:11 Last Changed Rev: 14677
Experimental Factor Name strain temperature
Experimental Factor Type ecotype growth_condition
Experimental Factor Term Source REF
Person Last Name Slotte
Person First Name Tanja
Person Mid Initials
Person Email Tanja.Slotte@ebc.uu.se
Person Phone +46184716421
Person Fax
Person Address Norbyv. 18D, Uppsala, NA, 75236, Sweden
Person Affiliation Dept of Evolution, Genomics and Systematics
Person Roles submitter
Person Roles Term Source REF The MGED Ontology
Quality Control Type biological_replicate dye_swap_quality_control
Quality Control Term Source REF The MGED Ontology The MGED Ontology
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2007-07-18
PubMed ID 17631524
Publication DOI 17631524
Publication Author List Slotte, Tanja; Holm, Karl; McIntyre, Lauren M.; Lagercrantz, Ulf; Lascoux, Martin
Publication Title Differential Expression of Genes Important for Adaptation in Capsella bursa-pastoris (Brassicaceae)
Publication Status journal_article
Publication Status Term Source REF The MGED Ontology
Experiment Description Differential expression between seedlings of early- and late-flowering Capsella bursa-pastoris, with and without vernalization treatment
Protocol Name P-CAGE-25558 P-CAGE-25554 P-CAGE-25555 P-CAGE-25559 P-CAGE-25556 P-CAGE-25561 P-CAGE-25560 P-CAGE-25563
Protocol Type
Protocol Description Approximately 50 seeds sterilized in 10% bleach for 10 min and rinsed in 70% ethanol and distilled water were sown on 0.8% agar plates with Murashige-Skoog medium (Duchefa, Haarlem, the Netherlands). Plates were kept in darkness in a refrigerator at 2.6ºC for 3 days before transfer to a growth chamber. Seeds were sterilized for 10 min in 10% bleach, followed by a 1 min rinse in 70% ethanol, and five washes using sterile water. Approximately 50 sterilized seeds were sown on each 0.8% agar plate with Murashige-Skoog medium (Duchefa, Haarlem, the Netherlands). Plates were placed in randomized order in a growth chamber under long day conditions (16 h light/8 h dark, 18:22ºC, 43% humidity, light intensity 250 μmol m-2 s-1). Approximately 50 seeds sterilized in 10% bleach for 10 min and rinsed in 70% ethanol and distilled water were sown on 0.8% agar plates with Murashige-Skoog medium (Duchefa, Haarlem, the Netherlands). Plates were kept in darkness in a refrigerator at 2.6ºC for 28 days before transfer to a growth chamber. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, California, USA), including DNase treatment using the RNase-free DNase Set (Qiagen), according to the manufacturers instructions.
cDNA synthesis and purification
First-strand cDNA synthesis was accomplished by adding, to a total of 5 μg of total RNA, 1 μl of 0.5 μg/μl PAGE-purified T7dT primer (TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTNN), 1 μl of ten times diluted Lucidea Universal Scorecard control mixes (GE healthcare Bio-Sciences Corp., Piscataway, New Jersey, USA) and Rnase-free water to a final volume of 11 μl. After incubation for 10 min at 70ºC the mix was quickly chilled on ice and the contents were collected by quick centrifugation. A total of 4 μl 5X First Strand Buffer (Invitrogen, Carlsbad, CA), 2 μl 0.1M DTT (Invitrogen) and 1 μl of 10mM dNTP solution (Invitrogen) was added, and the contents were incubated at 37ºC for 2 min prior to addition of 2 μl SuperScript III reverse transcriptase enzyme (Invitrogen). The mix was incubated at 37ºC for two hours for reverse transcription and subsequently placed on ice.
Second strand cDNA synthesis was done by adding a total of 91 μl of Rnase-free water, 30 μl 5X Second Strand Buffer (Invitrogen), 3 μl 10 mM dNTPs, 4 μl E. coli Polymerase I (Invitrogen) and 1 μl RNase H (Invitrogen) to each first-strand synthesis reaction and incubating the mix for 2 hours at 16ºC. To stop the reaction, 10 μl of 0.5 M EDTA was added.
To purify the cDNA, each cDNA synthesis reaction mix was transferred into an phase-lock gel tube (Eppendorf, Hamburg, Germany) and 160 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed thoroughly. Phase-lock tubes were centrifuged for 5 min at 16060 × g. Following centrifugation, the supernatant was collected and placed in a fresh tube. An equal volume of 5 M NH4OAC, 1 μl of linear acrylamide and 2.5 volumes of 99.5% ethanol were added. After mixing, the tubes were put on ice for 10 min and then centrifuged for 30 min at 16060 × g at 4ºC. The supernatant was removed and washed twice with 500 μl of 80% ethanol with 5 min centrifugation at 16060× g after each wash. The purified cDNA was air-dried and re-suspended in 10 μl of RNase-free water.
In vitro transcription
In vitro transcription was performed using Ambions Megascript T7 kit (Ambion Inc., Austin, Texas, USA). A total of 5 μl purified cDNA was used as starting material, and the reaction was set up according to the manufacturers instructions. In vitro transcription reactions were incubated at 37ºC overnight. The RNA was purified on an Rneasy column (Qiagen, Valencia, California, USA) as follows. A total of 80μl of RNase-free water, 350μl of Buffer RLT (Qiagen) with β-mercaptoethanol and 250 μl of 99.5% ethanol was added to the RNA and the sample was mixed by pipetting. The sample was transferred to an RNeasy mini spin column and centrifuged for 15 s at 16060× g, transferred to a new collection tube and washed twice with 500 μl of Buffer RPE (Qiagen). The first wash was followed by a 15 s centrifugation at 8000 × g and the second wash was followed by a 2 min centrifugation at 16060 × g. After additional centrifugation for 1 min at 16060 × g the column was transferred to a 1.5 ml collection tube and purified RNA was eluted twice in 30 μl RNase-free water. The concentration of the in vitro transcribed RNA (aRNA) was checked on a Nanodrop (Wilmington, Delaware, USA) and the integrity was checked on a 1% agarose gel.
Approximately 15 entire seedlings were pooled and RNA extraction conducted on the pool. One pool consisted of seedlings from a single ecotype and treatment grown on a single plate. Aminoallyl labeling of cDNA for microarrays
To 5 μg aRNA, 2 μl of random hexamer primers (3 mg/ml) were added and the final volume was brought up to 18.5 μl with RNase-free water. After a 10 min incubation at 70ºC, the mix was transferred to ice for 1 min and centrifuged briefly. The remaining steps were done at room temperature. A 50 × aminoallyl-dNTP mix containing 25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP and 10 mM amino-allyl-dUTP (Sigma-Aldrich, St Louis, Missouri, USA) was prepared. To each sample, 6 μl of First Strand Buffer (Invitrogen), 3 μl 0.1 M DTT (Invitrogen) and 0.6 μl 50 × aminoallyl-dNTP mix and 2 μl SuperScript III reverse transcriptase (Invitrogen) was added. The resulting mix was incubated at 42ºC overnight to produce first-strand cDNA with aminoallyl-dUTP incorporated. RNA was hydrolyzed by addition of 10 μl of 1 M NaOH and 10 μl of 0.5 M EDTA and incubation at 65ºC for 15 min. To neutralize the pH, 10 μl of 1 M HCl was added to each sample.
To remove unincorporated aminoallyl-dUTP and free amines, the aminoallyl-labeled cDNA was purified. The cDNA reaction was mixed with 300 μl Buffer PB (Qiagen) and transferred to a QIAquick column (Qiagen). The column was centrifuged at 16060 × g for 1 min after which the eluate was re-added and centrifugation repeated. Two washes were carried out using 650 μl phosphate wash buffer (5 mM KPO4, pH 8.0, 80% ethanol) with 1 min centrifugation at 16060 × g following each wash. After drying the membrane by 1 min centrifugation at 16060 × g, purified cDNA was eluted twice in 30 μl phosphate elution buffer (4 mM KPO4, pH 8.5). The sample was dried to completion in a vacuum centrifuge, and the aminoallyl-labeled cDNA was resuspended in 4.5 μl 0.1 M Na2CO3, pH 9.0. To each sample, 4.5 μl of Cy3 dye ester (GE Healthcare) was added and the reaction was incubated for 1 hour in the dark at room temperature. A total of 35 μl 100 mM NaOAc, pH 5.2, and 250 μl Buffer PB (Qiagen) was added, the mix was transferred to a Qiaquick spin column (Qiagen) and centrifuged at 16060 × g for 1 min. The eluate was re-added and the centrifugation repeated. A single wash using 650 μl Buffer PE (Qiagen) with 1 min centrifugation at 16060 × g was carried out. After a 1 min additional centrifugation at 16060 × g to dry the membrane the labeled cDNA was eluted twice in 30 μl Buffer EB (Qiagen). The labeled cDNA was analyzed on a Nanodrop to assess dye incorporation.
Aminoallyl labeling of cDNA for microarrays
To 5 μg aRNA, 2 μl of random hexamer primers (3 mg/ml) were added and the final volume was brought up to 18.5 μl with RNase-free water. After a 10 min incubation at 70ºC, the mix was transferred to ice for 1 min and centrifuged briefly. The remaining steps were done at room temperature. A 50 × aminoallyl-dNTP mix containing 25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP and 10 mM amino-allyl-dUTP (Sigma-Aldrich, St Louis, Missouri, USA) was prepared. To each sample, 6 μl of First Strand Buffer (Invitrogen), 3 μl 0.1 M DTT (Invitrogen) and 0.6 μl 50 × aminoallyl-dNTP mix and 2 μl SuperScript III reverse transcriptase (Invitrogen) was added. The resulting mix was incubated at 42ºC overnight to produce first-strand cDNA with aminoallyl-dUTP incorporated. RNA was hydrolyzed by addition of 10 μl of 1 M NaOH and 10 μl of 0.5 M EDTA and incubation at 65ºC for 15 min. To neutralize the pH, 10 μl of 1 M HCl was added to each sample.
To remove unincorporated aminoallyl-dUTP and free amines, the aminoallyl-labeled cDNA was purified. The cDNA reaction was mixed with 300 μl Buffer PB (Qiagen) and transferred to a QIAquick column (Qiagen). The column was centrifuged at 16060 × g for 1 min after which the eluate was re-added and centrifugation repeated. Two washes were carried out using 650 μl phosphate wash buffer (5 mM KPO4, pH 8.0, 80% ethanol) with 1 min centrifugation at 16060 × g following each wash. After drying the membrane by 1 min centrifugation at 16060 × g, purified cDNA was eluted twice in 30 μl phosphate elution buffer (4 mM KPO4, pH 8.5). The sample was dried to completion in a vacuum centrifuge, and the aminoallyl-labeled cDNA was resuspended in 4.5 μl 0.1 M Na2CO3, pH 9.0. To each sample, 4.5 μl of Cy5 dye ester (GE Healthcare) was added and the reaction was incubated for 1 hour in the dark at room temperature. A total of 35 μl 100 mM NaOAc, pH 5.2, and 250 μl Buffer PB (Qiagen) was added, the mix was transferred to a Qiaquick spin column (Qiagen) and centrifuged at 16060 × g for 1 min. The eluate was re-added and the centrifugation repeated. A single wash using 650 μl Buffer PE (Qiagen) with 1 min centrifugation at 16060 × g was carried out. After a 1 min additional centrifugation at 16060 × g to dry the membrane the labeled cDNA was eluted twice in 30 μl Buffer EB (Qiagen). The labeled cDNA was analyzed on a Nanodrop to assess dye incorporation.
Microarrays were scanned using an Axon 4000B scanner (Axon Instruments), at resolution 10 μM and 532 nm PMT values of 640-750 and 635 nm PMT values of 840-900. Image analysis was performed using 'Spot' from CSIRO: http://www.cmis.csiro.au/iap/Spot/spotoutput.htm
Protocol Parameters media;day temperature;night temperature;day humidity;light hours;night humidity;light source;light intensity; Extracted product;Amplification; Used Label;Amplification;Amount of nucleic acid labeled; Amount of nucleic acid labeled;Used Label;Amplification;
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF
SDRF File E-ATMX-22.sdrf.txt
Term Source Name ncbitax The MGED Ontology ArrayExpress The MGED Ontology mo EFO
Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version