Computational protocol: Detection and preliminary screening of the human gene expression profile for Hirschsprung's disease

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Protocol publication

[…] Sample labeling and array hybridization were performed using Agilent One-Color Microarray-Based Gene Expression Analysis (Agilent Technologies, Inc., Santa Clara, CA, USA), according the manufacturer's instructions. The total RNA from each sample was linearly amplified and labeled using Cy3-UTP (Enzo Life Sciences). The labeled cRNAs were purified using an RNeasy Mini kit (Qiagen). The concentration and specific activity levels of the labeled cRNAs (pmol Cy3/µg cRNA) were measured using the NanoDrop ND-1000 spectrophotometer. A total of 1 µg of each labeled cRNA was fragmented by adding 11 µl 10X blocking agent (Agilent Technologies, Inc.) and 2.2 µl of 25X fragmentation buffer (Agilent Technologies, Inc.). The mixture was then heated to 60°C for 30 min, and 55 µl 2X GE hybridization buffer (Agilent Technologies, Inc.) was added to dilute the labeled cRNA. A total of 100 µl hybridization solution was dispensed into a gasket slide, which was added to the gene expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent hybridization oven (Agilent Technologies, Inc.). The hybridized arrays were washed with Milli-Q water (Millipore, Billerica, MA, USA), fixed, and scanned using an Agilent DNA Microarray Scanner (G2505C; Agilent Technologies, Inc.).An Agilent Quick Amp Labeling kit (Agilent Technologies, Inc.) was used for the sample labeling. Hybridization was performed in Agilent SureHyb Hybridization Chambers (Agilent Technologies, Inc.). In GeneSpring GX v11.5.1, a normalized value is a relative number that comes from the ratio of the comparison between the raw value of the listed probe and that of the controls. Normalized values were calculated based on the normalization method applied to the data. Please refer to the technical note for information on how the control values were calculated. For one-color data, the four normalization methods (median, scale, quantile, and normalization to control genes) were used. For two-color data, each raw intensity value corresponding to the control channel was adjusted using a locally-weighted Lowess regression method. Each value in the signal channel was divided by the adjusted control value, resulting in the final normalized value. [...] The slides were washed with Milli-Q water and subsequently scanned using the Agilent DNA Microarray Scanner (Agilent Technologies, Inc.). Data was collected using Agilent Feature Extraction software (Agilent Technologies, Inc.). Normalization and data analysis were performed using Agilent GeneSpring GX v11.5.1 (Agilent Technologies, Inc.). The results were provided in the Gene Expression Profiling Data.xls file and Data Analysis Folder. Agilent Feature Extraction software v11.0.1.1 (Agilent Technologies, Inc.) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Inc.). Following quantile normalization of the raw data, genes for which a minimum of three out of the nine samples were statistically significant were selected for further data analysis. Genes that were differentially expressed in the two groups to a statistically significant degree were identified using volcano plot filtering. Hierarchical clustering was performed using Agilent GeneSpring GX software v11.5.1 (Agilent Technologies, Inc.). Gene ontology (GO) analysis and pathway analysis were performed using a standard enrichment computation method. The results were analyzed for three parameters: Molecular function, biological process and cellular components. The analyses were based on volcano plots, box plots, scatter plots, heat maps, hierarchical clustering, P-values, absolute fold change and GO assessed. Preliminary screening was used to determine the genes with statistically significant differences in expression levels in children with HSCR, in order to determine target genes for further verification and analysis step. […]

Pipeline specifications

Software tools GeneSpring GX, Agilent Feature Extraction
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Hirschsprung Disease, Colorectal Neoplasms