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Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.

ABSTRACT: Real-time quantitative polymerase chain reaction (qPCR) is a routine and robust approach for measuring gene expression. The stability of reference genes in qPCR is crucial for the accurate quantification of gene expression. To provide reliable reference genes for studying the transcriptional responses of locust muscles to hypobaric hypoxia, we first examined the gene expression stability of the frequently used housekeeping genes 18S, GAPDH, and ?-actin. However, the expression of these three housekeeping genes was influenced by hypobaric hypoxia. Consequently, we identified five novel candidate reference genes from the locust microarray data. The gene expression stability of the five candidates, together with the three classical housekeeping genes, were evaluated using two distinct algorithms implemented in geNorm and NormFinder. GeNorm identified Ach (acetyl-CoA hydrolase) and Pgp (phosphoglycolate phosphatase-like) as the most stable genes and NormFinder further distinguished Ach as the most stable one. The validity of Ach as a reference gene was confirmed through comparison with 18S. This study exemplifies the necessity of validating reference genes before their application and the feasibility of identifying condition-specific reference genes from large-scale gene expression data.

SUBMITTER: Zhao DJ

PROVIDER: S-EPMC3642161 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.

Zhao De-Jian DJ Guo Kun K Kang Le L

FEBS open bio 20120813

Real-time quantitative polymerase chain reaction (qPCR) is a routine and robust approach for measuring gene expression. The stability of reference genes in qPCR is crucial for the accurate quantification of gene expression. To provide reliable reference genes for studying the transcriptional responses of locust muscles to hypobaric hypoxia, we first examined the gene expression stability of the frequently used housekeeping genes 18S, GAPDH, and β-actin. However, the expression of these three hou ...[more]

PMID: 23650605

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Project description:Leptin and hypoxia are pro-fibrotic factors involved in fibrogenesis, however, the gene expression profiles remain to be fully elucidated. The aim of the present study was to investigate the regulatory roles of leptin and hypoxia on the L929 mouse fibroblast cell line. The cells were assigned to a normoxia, normoxia with leptin, hypoxia, and hypoxia with leptin group. The cDNA expression was detected using an Agilent mRNA array platform. The differentially expressed genes (DEGs) in response to leptin and hypoxia were identified using reverse transcription?quantitative polymerase chain reaction analysis, followed by clustering analysis, Gene Ontology analysis and pathway analysis. As a result, 54, 1,507 and 1,502 DEGs were found in response to leptin, hypoxia and the two combined, respectively, among which 52 (96.30%), 467 (30.99%) and 495 (32.96%) of the DEGs were downregulated. The most significant functional terms in response to leptin were meiosis I for biological process (P=0.0041) and synaptonemal complex for cell component (P=0.0013). Only one significant pathway responded to leptin, which was axon guidance (P=0.029). Flow cytometry confirmed that leptin promoted L929 cell proliferation. The most significant functional terms in response to hypoxia were ion binding for molecular function (P=7.8621E?05), glucose metabolic process for biological process (P=0.0008) and cell projection part for cell component (P=0.003). There were 12 pathways, which significantly responded to hypoxia (P<0.05) and the pathway with the highest significance was the chemokine signaling pathway (P=0.0001), which comprised 28 genes, including C?C motif ligand (CCL)1, C?X?C motif ligand (CXCL)9, CXCL10, son of sevenless homolog 1, AKT serine/threonine kinase 2, Rho?associated protein kinase 1, vav guanine nucleotide exchange factor 1, CCL17, arrestin ?1 and C?C motif chemokine receptor 2. In conclusion, the present study showed that leptin and hypoxia altered the pro?les of gene expression in L929 cells. These findings not only extend the cell spectrum of leptin on cell proliferation, but also improve current understanding of hypoxia in fibroblast cells.

Dataset Information

Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.

Publications

Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.

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