Computational protocol: Understanding the molecular aspects of oriental obesity pattern differentiation using DNA microarray

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Protocol publication

[…] Total RNA of the PMBCs was extracted using TRI reagent (Ambion, Austin, TX, USA) and the RNeasy mini kit (Qiagen, Hilden, Germany) following the reagent and kit manufacturer’s instructions, respectively. The yield of RNA ranged from 5.02 to 15.37 μg with an average of 8.85 μg. The integrity of extracted RNA was verified by gel electrophoresis. DNA microarray of the samples and subsequent analysis of the data were performed as described previously []. Briefly, 5 μg of total RNA was reverse transcribed for generation of double-stranded cDNA (dscDNA) using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Reactions were terminated by addition of EDTA followed by RNase A treatment. Samples were then subjected to ethanol-precipitation and finally rehydrated to make the stock solution of dscDNA at a concentration of 250 ng/μl. Next, 1 μg dscDNA was labeled with Cy3-conjugated random 9-mer (TriLink Biotechnologies, San Diego, CA, USA) using Klenow fragment (NEB, Beverly, MA, USA); the labeled samples were then subjected to isopropanol precipitation. Four micrograms of Cy3-labeled DNA (containing sample tracking control and alignment oligo) was then hybridized to NimbleGen, 12-plex, human microarray slides (Human Gene Expression 12 × 135 K Microarray, NimbleGene, Madison, WI, USA) for 18 h at 42 °C using the NimbleGen Hybridization system (NimbleGen). Subsequently, the array slides were washed by vigorous agitation in 1 × SSC + 0.1 % SDS for 5 min at 55 °C and in 0.1 × SSC + 0.1 % SDS for 5 min at room temperature. The slides were then rinsed with distilled water and dried by centrifugation. The array images were captured using an InnoScan 900 Series Microarray Scanner (Innopsys, Carbonne, France) and the signals extracted from the scanned images were then imported into NimbleScan software (version 2.5, Nimblegen) for grid alignment and analysis of gene expression data. Expression data were normalized using a quantile normalization method [] and Robust Multichip Average (RMA) algorithms []. Gene ontology (GO) analysis was performed using the software toolkit provided in the web-accessible Database for Annotation, Visualization, and Integrated Discovery (DAVID) programs (http://niaid.abcc.ncifcrf.gov/home.jsp) []. For this analysis, the selected gene list was uploaded into the program and the gene list of Nimblegen microarray chip was used as the background. For clustering analysis, data from microarray was uploaded to the Cluster program and cluster analysis was performed by applying parameters of interest, as described previously []. Clustered data were visualized using the TreeView program. [...] Results are expressed as the mean ± standard deviation (SD). All statistical analyses were performed using SPSS software (Version 20, SPSS Inc, Chicago, IL, USA). One-way ANOVA was used for comparison of differences among the three oriental obesity patterns. Independent student’s t test was used for comparison of differences between two groups. Correlations between clinical parameters and the level of gene expression were assessed by Pearson’s correlation test. To determine correlation values, heat map analyses were performed using PermutMatrix software (Version 1.0.3 EN, PermutMatrix, Montpellier, France). P value <0.05 was considered statistically significant. As mentioned above, gene ontology (GO) term enrichment was analyzed using tools available on the DAVID website [] and data from ‘GOTERM_BP_ALL’ database are presented in Table . […]

Pipeline specifications

Software tools DAVID, TreeView, PermutMatrix
Applications Gene expression microarray analysis, Transcriptome data visualization
Organisms Homo sapiens
Diseases Foodborne Diseases, Hematologic Diseases, Metabolic Diseases, Splenic Neoplasms