MeJA-induced gene expression in rice plants

ABSTRACT: To understand signal transduction mechanism by MeJA in rice, we have analyzed transcription profile with 60K Rice Whole Genome Microarray after MeJA treatment. Gene transcripts were extracted from ten individual rice plants treated with 100 uM MeJA for 6 hrs. RNA samples from these plants were used to generate cyanine-3 (Cy3) and Cy5-labeled complementary DNA (cDNA) probes, which were then hybridized to the microarray. Each data set was obtained from three biological repeats independently. Preparation of fluorescence labeled probes and microarray hybridization was performed as procedures provided by Genisphere 3DNA Array Detection Array 50 kit (v. 2, Genisphere, Hatfield, PA). The microarray was scanned with Genepix 4000B (Axon Instruments, Union City, CA) and the quality of the chip data was analyzed with statistical R language and sma package in Bioconductor project (http://www.bioconductor.org/) implemented on Linux platform. Noncorrelation of signal and background intensities were confirmed by plotting base 2 log background intensity in x axis and base 2 log intensity subtracted with background intensity in y axis. Prior to normalization, normal distribution of Cy3 and Cy5 intensities were tested by qqplot function in R statistical language. The spatial effects on the chip during the hybridization process were checked with spatial func in sma package. The variance difference between Cy3 and Cy5 intensities within microarray was tested by the Student's t test under the assumptions firstly uniform and then nonuniform variances. The ANOVA difference of signal intensities between microarrays was performed by one-way ANOVA. Block-by-block Lowess normalization (Yang et al., 2002) and multivariate statistics such as clustering, principal component analysis, multidimensional scaling, etc. were analyzed with Acuity 3.1 (Axon Instruments). Spots with flag of 0 and a diameter greater than 51 pixel size were used for the analysis. Cy3 significant spots were determined when its log ratio is less than –0.67 [2**(–0.67) = 1.6-fold decrease] and its background subtracted intensity is higher than 500. On the contrary, Cy5 significant spots was determined when its log ratio is greater than 0.67 [2**0.67 = 1.6-fold increase] and its intensity is higher than 500. A preliminary microarray experiment using nontreated wild-type RNA labeled Cy3 and Cy5, respectively, gave less than 0.5% false positive signals in the analysis. To assess the reproducibility of the microarray analysis, we repeated the experiment three times with independently prepared total RNA

ORGANISM(S): Oryza sativa

SUBMITTER: Ju-Seok Seo 

PROVIDER: E-GEOD-22757 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress