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Arabidopsis thaliana seedlings:early light regulated gene expression in Ler wildtype versus eid3 mutant

ABSTRACT: Transcriptional profiling of Arabidopsis thaliana Ler wildtype and eid3 (empfindlicher im dunkelroten Licht 3) mutant seedlings in darkness and 45 min after a red-light pulse. Arabidopsis thaliana Ler wildtype and eid3 mutant seedlings were grown on 1/2 MS Agar plates covered with filter paper for 4 days in darkness after induction of germination with 2 h red light. Samples were either treated with 2 min red light (30 µmol/m2s) or kept in darkness and harvested after additional 45 min in darkness. 3 biological replicas were used for each of the 4 experimental conditions.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Cornelia Klose

PROVIDER: E-GEOD-33856 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Treatment of the seeds. The Solanum lycopersicum var. Money Maker wild type (MM) and fri1-1 mutant seeds were incubated on distilled-water saturated cotton wool in clear plastic boxes wrapped in black plastic sheets in darkness during 3 hours at 25°C. After that, the seeds were treated as follows: a) 24 hr of continuous irradiation with far-red light, FRc; b) 24 hr of FRc follow by a 10 min red light pulse (Rp), FRc+Rp; and c) complete darkness. After the light treatments, the seeds were incubated in darkness (25°C) until sampling. Red light (35umol/m2sec) was provided by 40W fluorescent lamps (Phillips 40/15) in combination with a red translucid acrylic. FRc light (100 umol/m2sec) was provided by a set of 150W incandescent internal reflector lamps (Phillips) in combination with a red acetate filter, six 2mm thick blue acrylic filters Paolini 2031 and 10cm water filter. Total RNA isolation. The seeds were grinded in liquid nitrogen and extracted with two volumes of extraction buffer (1% SDS, 6% p-aminosalycilic acid, 1% NaCl, 6% water-saturated phenol and 2% 2-mercaptoethanol) and two volumes of acid phenol:chloroform solution. The aqueous phase was sequentially extracted with one volume of acid phenol:chloroform solution and one volume of chloroform. The nucleic acids in the aqueous phase were precipitated with 0.15 volumes of 3M NaCl and 2.5 volumes of 100% ethanol at –80°C for 20 minutes. After centrifugation, the pellet was washed with 70% ethanol, resuspended in RNase-free water and precipitated with one volume of 4M LiCl over night at 4°C. The LiCl was removed after centrifugation and the pellet was rinsed with 2M LiCl and resuspended in RNase-free water. The total RNA was treated with RNase-free DNase (Promega) and cleaned up with mini spin columns (RNeasy Plant Mini Kit, Qiagen). The mRNA was amplificated with Message Amp II aRNA Amplification Kit (Ambion) and concentrated by vacumm centrifugation. Keywords: Direct comparison 21 hybs total

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Arabidopsis thaliana seedlings:early light regulated gene expression in Ler wildtype versus eid3 mutant

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