Computational protocol: S100A8 and S100A9 Are Associated with Doxorubicin-Induced Cardiotoxicity in the Heart of Diabetic Mice

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Protocol publication

[…] Forty micrograms of frozen ventricular tissues were minced and homogenized in ice-cold TriReagent (Molecular Research Center, USA). The tissue homogenate was then processed to separation of the aqueous and organic phases. The RNA in the aqueous phase was precipitated by addition of isopropanol followed by washing with 75% ethanol. The extracted RNA pellets were then re-suspended in DEPC-treated water. RNA concentrations were quantified in triplicate using the optical density (OD) at 260 nm. In this study, the technique of microarray analysis was adopted to examine the changes of whole transcriptional profile in response to the administration of DOX in diabetic heart. RNA samples extracted from the ventricular tissues of mice in db/dbCON and db/dbDOX groups 5 days after the DOX administration were used in the microarray analysis. RNA samples (3 μg) from the hearts of two mice in each group were pooled to generate four biological replicates in db/dbCON and db/dbDOX groups. The RNA quantity and quality were assessed before microarray analysis. RNA quantity was detected by NanoDrop 1000 Spectrophotometer. The purity of RNA was assured by examining the OD260/280 ratio. The RNA integrity was assessed by Agilent 2100 bioanalyzer by following the manufacturer's instruction. In the present study, our RNA samples used for array hybridization showed satisfactory integrity with intact bands corresponding to 18 and 28 S ribosomal RNA and the RNA Integrity Number (RIN) was >7.Microarray analysis was performed using the Agilent Service Platform with Agilent two-color mouse 4 × 44 k microarray slides. Samples from the DOX-treated diabetic mice (db/dbDOX) were labeled by Cy5 dye (red channel) whereas samples from diabetic control mice (db/dbCON) were labeled by Cy3 dye (green channel). Five hundred nanograms of Cy3-labeled and Cy5-labeled cRNA were mixed and incubated with the Agilent microarray slide (G2519F) for 17 h at 65°C in the dark. Slide was washed and scanned using an Agilent DNA microarray scanner. Raw data were obtained using Agilent's Feature Extraction 10.7 Software. Further analysis of the raw data was performed by ArrayTrack comprehensive R—and Bioconductor-based web service for microarray data analysis. Several normalizations were performed for the pre-processing of the raw data and these included background correction by subtraction method, removal of dye bias by lowess normalization and multiple testing correction by BH adjusted P-values for the Benjamini and Hochberg step-up FDR controlling procedure (Benjamini and Hochberg, ). Gene expression values were calculated by log base 2 ratio of red channel intensity (mean), and green channel intensity (mean). Functional pathways of highly regulated genes were analyzed by MetaCore® (Version 6) from GeneGo Inc. […]

Pipeline specifications

Software tools ArrayTrack, MetaCore
Application Gene expression microarray analysis
Organisms Homo sapiens, Mus musculus
Diseases Diabetes Mellitus, Heart Diseases, Cardiomyopathies, Neoplasms, Diabetic Cardiomyopathies, Drug Overdose
Chemicals Doxorubicin, Ketamine, Xylazine