Computational protocol: Gene Expression Signature of DMBA-Induced Hamster Buccal Pouch Carcinomas: Modulation by Chlorophyllin and Ellagic Acid

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Protocol publication

[…] Total RNA was isolated from the buccal pouch of two individual hamsters in each experimental group using Trizol as described previously . Isolated RNA was then purified using RNAeasy columns (Qiagen GmbH, Hilden, Germany) and treated with ribonuclease free DNaseI to remove any contaminating DNA. RNA was quantitated by NanoDrop ND-1000 (Thermo Scientific, Wilmington DE) and the quantity and integrity was established by resolving on 0.8% formaldehyde agarose gels.Microarray experiments were performed in replicates using Agilent Whole Rat Genome Oligo Microarray Kit, 4×44 k. As genome sequence information of hamsters are relatively limited, rodent microarrays such as mouse or rat DNA microarrays are used for the transcriptome profiling of Syrian hamster tissues and Chinese hamster ovary cells . For the microarray labeling reaction, 200 ng of isolated RNA from each sample was used. Labeling was done using the Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA) as per the manufacturer's protocol. Briefly, using affinity script-RT oligo dT promoter primer, cDNA was generated and from the cDNA, labeled cRNA was generated via an in vitro transcription reaction using T7 RNA polymerase and Cy5 (DMBA, DMBA+chlorophyllin, DMBA+ellagic acid) or Cy3 (control) CTP. Labeled cRNA (825 ng) from each sample was used for hybridization in the following combinations- DMBA (Cy5) and control (Cy3), DMBA+chlorophyllin (Cy5) and control (Cy3) and DMBA+ellagic acid (Cy5) and control (Cy3). Hybridization was performed for 17 h, rotating at a speed of 10 rpm at 65°C in a hybridization oven (Agilent Technologies).Microarray data analysis was done by using Limma package in R-Bioconductor. Mean foreground and median background expression values given by Agilent feature extraction Software was used for analysis. Background subtraction was done using “subtract” method in Limma, dye normalization was done by LOESS algorithm, and between array normalization was done by “quantile” method. Imfit and eBayes (Empirical Bayes method) were used to find differentially regulated genes. P value correction was done using Benjamini and Hochberg method. P value cutoff was made as 0.05. Gene enrichment analysis of the differentially regulated genes with two-fold change cut off was done using GSEA Pre Ranked Tool. […]

Pipeline specifications

Software tools limma, Agilent Feature Extraction
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Carcinoma, Leukoedema, Oral, Neoplasms
Chemicals Ellagic Acid, 9,10-Dimethyl-1,2-benzanthracene