Comment[GEAAccession]	E-GEAD-316
MAGE-TAB Version	1.1
Investigation Title	Expression profiling of the response of Arabidopsis thaliana in mature feeding sites formed by Meloidogyne incognita.
Experiment Description	Meloidogyne incognita is an obligate endoparasite of plants. This nematode inhabits the soil and infects the root systems of susceptible plants. The nematodes migrate within the root and establish a feeding site from plant xylem parenchymal cells which are stimulated by the nematode to become multinucleate giant cells surrounded by hypertrophic tissue that forms a gall. These giant cells act as transfer cells and supply the parasitic nematode with all of its nutritional requirements during the course of its 3-8 week lifecycle. Little is known about the molecular changes induced in the feeding site by the nematode to facilitate this process. Microarray analysis was therefore performed using whole genome Arabidopsis microarrays with RNA obtained from feeding sites formed by M. incognita in Arabidopsis. Feeding sites were extracted from roots at 21 days post infection and the gene expression profile compared to that in uninfected roots.
Experimental Design	disease state design
Experimental Factor Name	treatment
Experimental Factor Type	treatment
Person Last Name	Fuller
Person First Name	Victoria
Person Affiliation	Plant Nematology Laboratory, Centre for Plant Sciences, University of Leeds
Person Roles	submitter
Public Release Date	2019-09-17
PubMed ID	20507524
Protocol Name	P-GEAD-210	P-GEAD-211	P-GEAD-212	P-GEAD-213	P-GEAD-214	P-GEAD-215	P-GEAD-216	P-GEAD-217
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol	growth protocol	treatment protocol
Protocol Description	Several 100 sections of uninfected roots from different control plants were pooled over a period of approximately 12 months. Several 100 galls from different plants were pooled over a period of approximately 12 months.	Biological material was stored in RNALater (Ambion) at -80 degree C. RNA was extracted using Qiagen RNeasy Plant Mini Kit, according to the manufacturer's protocol	RNA was amplified and labeled with Cy5 or Cy3 using Amino Allyl MessageAmp method (Ambion)	A hybridisation mix was prepared with 3-6 ug each of Cy3- and Cy5-labelled aRNA and 4 ug of sonicated salmon sperm DNA (Stratagene, La Jolla, U.S.A.) in ArrayHyb Low Temp hybridization buffer (Sigma) to a final volume of 330 ul. The mix was vortexed, incubated at 60 degree C for 3 min and ice quenched for 5 min before being centrifuged briefly and pipetted onto a microarray within a Gene Frame (Abgene, Epsom, UK). The slides were incubated at 50 degree C for 24 h within a Genetix hybridisation chamber.	Arrays were scanned with a Genepix 4000B scanner at a resolution of 10 um. A minimum of four PMT values starting with the lowest settings was used for each slide, and red and green channels were scanned simultaneously. TIFF images were quantified with ArrayVision (vs. 8, Imaging Research Inc., Ontario, Canada) using median density values of the fluorescence of gene elements and local median background intensity.	Artefact-removed median density values of the fluorescence of arrayed gene elements were measured using the iterative circle method. Background fluorescence was measured as local median background intensity and subtracted from signal spot intensities. 'Landing light' orientation spots of pre-bound Cy3 at a corner of each block were excluded from analysis. Background-subtracted median fluorescence values were normalised using the LOWESS algorithm by print-tip group in the ArrayVision programme, using an f value of 20%. Data for each microarray replicate was exported to Microsoft EXCEL and merged with block, column and row identifiers to provide full locus information for each spot on the array using Microsoft Access. This data was exported back into EXCEL to create a single file containing the normalised log10 ratios for all spots. Gene elements with fluorescence <1.2 local background fluorescence in one or both channels were removed. Further data processing was performed using MadScan software (Microarray Data Suite of Computed Analysis; www.madtools.org; Le Meur et al., 2004). All arrays were scaled to the same median absolute deviation. Removal of inconsistent, outlying data replicates between arrays was achieved by three iterative passes through the Zmad Test.	Four sterile Arabidopsis seeds were sown on Gamborg's B5 medium (Sigma) in square petri dishes (Sterilin). These were placed in a Sanyo Environmental Test Chamber under controlled conditions (16h light/8h dark cycle, average light intensity of 67 umols-1m-2). Plants were incubated at 24 degree C and plates were arranged according to a randomised block design and held at approximately 70 degree C to facilitate downward growth of the roots.	Roots systems were mock-inoculated with 35 ul sterile tap water at 3 points per root system and covered with GF/A paper for 24 h.
SDRF File	E-GEAD-316.sdrf.txt
Comment[Number of channel]	dual-channel
Comment[Array Design REF]	A-GEOD-10055
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX23
Comment[BioProject]	PRJDB8783
Comment[CIBEX Accept Date]	2007-05-23
Comment[CIBEX Public Release Date]	2007-05-23
Comment[Last Update Date]	2019-09-17