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Appressorium induction by cyclic AMP in Magnaporthe grisea

ABSTRACT: This series examines differential gene expression during appressorium formation induced by exogeous cyclic AMP in the rice blast fungus Magnaporthe grisea. RNA was extracted from spores germinated on hydrophilic (non-inductive) side of GelBond as well as from spores elaborating appressia on hydrophilic surfaces in the presence of cAMP after 9 hrs incubation. RNA from two biological replications of each treatment was pooled in equal amounts and labeled with both cy3 and cy5 dyes using the Agilent Technologies Low Input Linear Amplification Kit. Hybridizations were performed on Agilent Technologies whole genome oligo rice blast arrays (product G4137B) using manufacturer prescribed protocols and reagents in an reference design in which each treatment was paired with every other. This series contains a total of 4 hybridizations; each treatment was used in 4 hybridizations (2 with Cy3 and 2 Cy5). Spot fluorescence was normalized using Lowess within and between microarray slides in Bioconductor. Keywords: Cell development

ORGANISM(S): Pyricularia grisea

PROVIDER: GSE10173 | GEO | 2008/01/15

SECONDARY ACCESSION(S): PRJNA108379

REPOSITORIES: GEO

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Project description:One µg of total RNA from either an individual rat or from a pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA). For day/night comparisons, two types of complementing hybridizations were performed: hepatic RNA from individual day rats was hybridized against a pool made up of RNA from 12 hour offset night rats, and RNA from individual night rats was hybridized against a pool made up of RNA from 12-hour offset day rats. Specifically, hepatic RNA samples from three individual day rats collected ten hours after light on (CT10) were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six night rats collected ten hrs after lights off (CT22); and RNA samples from three individual night rats collected at CT22 were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six day rats collected at CT10. This was replicated in a second study for a total of 24 hybridizations of 12 hour offset samples. For time of day comparisons, equal aliquots of RNA from the livers of six rats collected at each of four different times of the circadian day (CT4, CT10, C16 and CT22) were pooled. The two replicate studies thus resulted in 8 pools (two replicate pools/time point times four time points); each pool was hybridized against a Universal Rat Reference RNA Standard (Stratagene, La Jolla, CA). Keywords: time-course

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Appressorium induction by cyclic AMP in Magnaporthe grisea

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