Abstract
BackgroundWith the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., β-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions.Methodology/Principal FindingsTherefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization.Conclusion/SignificanceData are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.
Highlights
The obesity epidemic has led to a plethora of investigations examining mechanisms that regulate adipocyte differentiation and function as well as the role adipose tissue plays in the development of insulin resistance, diabetes and heart disease
Our report presents empirical evidence demonstrating that inappropriate reference gene selection for quantitative reverse transcription polymerase chain reaction (qRT-PCR) normalization can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude
We demonstrate that many commonly used reference genes fluctuate in a condition-specific manner beyond the limits of suitability when normalizing for target gene expression using qRT-PCR
Summary
The obesity epidemic has led to a plethora of investigations examining mechanisms that regulate adipocyte differentiation and function as well as the role adipose tissue plays in the development of insulin resistance, diabetes and heart disease. While earlier work with conventional methodology provided qualitative assessment of mRNA abundance, the quantitative nature of real time qRT-PCR affords a measure of sensitivity that is suited for reliable detection of 2-fold changes in gene expression over dynamic ranges of starting material [2,5] This methodology comes with a price, as increased sensitivity of qRT-PCR along with inherent variability in biological systems, experimental and extraction protocol disparity, as well as differences in reverse transcription and PCR efficiencies makes normalization of realtime data an absolute requirement for accurate data interpretation regarding genes of interest [5,6,7,8]. Historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., b-actin, GAPDH, a-tubulin) are differentially regulated across multiple tissue types and experimental conditions
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