Validation of suitable housekeeping genes for 3T3-L1 derived adipocytes cultured in obesity mimicking conditions and RAW 264.7 macrophage cells lines in hypoxic and normoxic conditions. (January 2019)

• Record Type: Journal Article
• Authors: Anbazhagan, Akshayavardhani; Sridharan, Preethi; Pratheesh, Pooja
• Is Part Of: Biocatalysis and agricultural biotechnology. Number 17(2019)
• Page Start: 620
• Page End: 627
• Publication Date: 2019-01
• Subjects: Agricultural biotechnology -- Periodicals; Enzymes -- Biotechnology -- Periodicals; 660.6
• DOI: 10.1016/j.bcab.2019.01.006
• Languages: English
• ISSNs: 1878-8181

Abstract

Abstract: Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) is the most commonly used method for gene expression analysis, and reliable results depend on proper normalization by stable reference genes. Adipose tissue hypoxia is the new concept in understanding the major disorders associated with obesity and may facilitate cellular mechanisms associated with obesity like chronic inflammation, macrophage infiltration, adiponectin reduction, leptinelevation, adipocyte death, ER stress and mitochondrial dysfunction. The hypoxic conditions in the obese adipose tissue lead to differential expression of genes in the resident cells of adipose tissues including macrophages andhence reliable normalization of RT-qPCR data becomes crucial. In this study, the expression stability of 4 most commonly used candidate reference genes, ACTB, B2M, HMBS and 18SrRNA were evaluated in murine adipocytesand macrophage cell lines, in presence of high free fatty acids, under hypoxia and normoxia conditions. The results were analyzed by manually calculating fold difference using 2−ΔΔCt and validated by using two algorithms, NormFinder, and Bestkeeper. Our results showed HMBS and B2M as the most stably expressed genes in both cell lines under obesity mimicking conditions (in presence of high amount of FFAs and hypoxia). By contrast, ACTB and 18S rRNA were the least stable genes under the tested conditions. Furthermore, the expression levels of FABP4 andPPAR-γ (for adipocytes) and MCP-1 and IL-6 (for macrophages) were profiled in these cells to validate the selected reference genes. Our data provides a basis for gene expression analysis in future studies related to inflammation in hypoxia related obesity pathogenesis. Highlights: Many housekeeping genes are modulated under differential environmental conditions including hypoxia and obesity. Robust housekeeping genes are needed as reference for studying gene expression profile in various physiological and pathological conditions. Through this work, we studied the expression profile of four commonly used housekeeping genes namely ACTB, 18S rRNA, HMBS, and B2M in hypoxia associated obesity like conditions. We show that HMBS and B2M are stably expressed under hypoxia associated obesity like conditions and could be considered as internal controls for related studies in future.