Investigation Title Transcription profiling of the1, prc1, prc1_the1 and prc1_the2 gene knockout Arabidopsis dark-grown hypocotyls to identify genes involved in cell wall integrity signalling
Comment[Submitted Name] Identification of genes involved in cell wall integrity signalling in Arabidopsis dark-grown hypocotyls. Transcriptome Analysis of prc1 or the1 and prc1_the1
Experimental Design individual_genetic_characteristics_design transcription profiling by array
Experimental Design Term Source REF EFO
Comment[ArrayExpressReleaseDate] 2007-03-31
Comment[AEMIAMESCORE] 4
Comment[ArrayExpressAccession] E-MEXP-638
Comment[MAGETAB TimeStamp_Version] 2010-08-11 15:36:33 Last Changed Rev: 13058
Experimental Factor Name genotype strain
Experimental Factor Type genotype strain_or_line
Experimental Factor Term Source REF
Person Last Name samira
Person First Name elftieh
Person Mid Initials
Person Email elftieh@evry.inra.fr
Person Phone +33 1 60 87 45 20
Person Fax
Person Address 2 rue Gaston crémieux CP5708
Person Affiliation research ministery
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 2007-03-31
PubMed ID
Publication DOI
Publication Author List
Publication Title
Publication Status
Publication Status Term Source REF
Experiment Description Inhibition of cellulose synthesis by chemical inhibitors or in a mutant background leads to rapid inhibition of cell elongation. This inhibition appears to be an active process, which involves feedback signalling from the cell wall. We have isolated two loci THE1 and THE2, which are identified by mutations that partially suppress the dark-grown hypocotyl phenotype in a mutant background for cellulose synthase catalytic subunit CESA6_PROCUSTE1. THE1 encodes a receptor kinase and may play a role in this feedback signalling process. To identify genes that are regulated by THE1 and THE2, we compared the transcript profiles of 5 day-old dark-grown seedlings of the1-1_prc1-1 with prc1-1 ; the1-3_prc1-8 with prc1-8 ; prc1-8 or the1-3 with WS and the2-1_prc1-1 with prc1-1.
Protocol Name P-MEXP-22098 P-MEXP-21694 P-MEXP-8894 P-MEXP-8893 P-MEXP-8895 P-MEXP-18229
Protocol Type grow nucleic_acid_extraction labeling labeling hybridization feature_extraction
Protocol Description Media : Arabidopsis medium Somerville&Estelle 1990 (0% sucrose)
hygrometry : 70%
Temperature : 20°C
Light : 4h Flash but none during growth (prelevment under green light) http://www1.qiagen.com/literature/handbooks/PDF/RNAStabilizationAndPurification/FromAnimalAndPlantTissuesBacteriaYeastAndFungi/RNY_Mini/1016272HBRNY_062001WW.pdf 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN). 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN). Slide preparation In a Coplin jar, prepare 50 ml of pre-hybridization solution :
SSC 20 X 12,5 ml
BSA 10 % 5 ml
SDS 10 % 0,5 ml
H2O 32 ml
Pre-heat this solution at 42 °C and préhybridize slides at 42 °C for at least 1 hour.
Wash the slides in milli-Q water, and then isopropanol.
Blow-dry the slides.
THE SLIDES MUST BE USED WITHIN 1 HOUR!
Hybridization
Prepare 2X hybridation mix (35 µl per slide) :
SSC 20x 50 µl
SDS 10% 2 µl
Formamide 50 µl
Denature the probe 1 mn at 95°C and keep on ice.
Add one volume of 2X hybridation mix, mix and centrifuge.
Put the slide in the Corning hybridization chamber, and place a cover slip on it.
Pipet the probe between the slide and the cover slip.
Put 10 µl of water in the two holes and close the chamber.
Incubate in a water bath at 42°C over-night (16h)
Washes
Soak in 2X SSC, 0,1% SDS at 42°C to remove the cover slip, then wash in :
- 2X SSC, 0,1% SDS (42°C) 4 min
- 1X SSC 4 min
- 0,2X SSC 4 min
- 0,05X SSC 1-4 min
Blow dry the slide or centrifuge for 2 min at 2000 rpm.
Store in a dry and dark place until scanning. ll the slides for a same project are scanned at a constant PMT power (i.e. 650V for 635nm and 620V for 532nm).
Protocol Parameters max temperature;min temperature; Amplification;Extracted product; Amount of nucleic acid labeled;Label used;Amplification; Amount of nucleic acid labeled;Label used;Amplification; temperature;Volume;time;Quantity of label target used;Chamber type;
Protocol Hardware GenePix 4000A [Axon Instruments]
Protocol Software GenePix Pro [Axon Instruments]
Protocol Contact
Protocol Term Source REF The MGED Ontology
SDRF File E-MEXP-638.sdrf.txt
Term Source Name mo ncbitax po The MGED Ontology ArrayExpress EFO The MGED Ontology
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.plantontology.org http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php
Term Source Version