Computational protocol: Investigation of TGFβ1-Induced Long Noncoding RNAs in Endothelial Cells

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Protocol publication

[…] Total RNA was isolated using TRIzol™ (Invitrogen) reagent and quantified with the NanoDrop ND-1000 spectrophotometer. RNA integrity was confirmed by standard denaturing agarose gel electrophoresis. The expression profile of 30,584 human lncRNAs and 26,106 protein-coding transcripts was conducted with the Arraystar Human LncRNA Microarray V3.0. Sample labeling and array hybridization were performed on the Agilent Array platform. Briefly, total RNA from each sample was amplified and transcribed into fluorescent cRNA (Arraystar Flash RNA Labeling Kit, Arraystar) before 1 μg of each labeled cRNA was hybridized onto the microarray slide. The hybridized arrays were washed, fixed, and scanned with the Agilent DNA Microarray Scanner (Product# G2505C). The acquired array images were analyzed with the Agilent Feature Extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed with the GeneSpring GX v11.5.1 software package (Agilent Technologies). P values for the differentially expressed genes were determined with the t-test and adjusted for multiple testing with the Benjamini Hochberg method to minimize the false discovery rate. Volcano plot filtering, set at a threshold of ≥2.0-fold, was used to screen for lncRNAs and mRNAs that exhibited significantly different (P < 0.05; unpaired t-test) expression levels in the two study groups. Pathway analysis was based on the current Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene ontology (GO) analysis was performed with the topGO package of bioconductor system. […]

Pipeline specifications

Software tools Agilent Feature Extraction, GeneSpring GX, TopGO
Databases KEGG
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Cardiovascular Diseases, Neoplasms