Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes

Abstract

Background: With 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 findings: Therefore, 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/significance: Data 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.

Figure 1. Gain of statistical significance with inappropriate reference gene selection.

Total RNA was isolated over time from density-arrested 3T3-L1 preadipocytes treated with 1 nM TNFα and assessed for reference and target gene expression by qRT-PCR. Fold changes in reference genes that fell within (A) and outside (B) the ΔC_T ≤ +/−0.5 limits of suitability. Fold changes in Dusp10 gene expression without (C) and with normalization (D) to 18S, the geometric mean of three reference genes or β-actin. Asterisks indicates significant differences between treated and untreated samples (p<0.05).

Figure 2. Loss of statistical significance with inappropriate reference gene selection.

Total RNA was isolated over time from density-arrested 3T3-L1 preadipocytes treated with 300 µM H₂O₂ and assessed for reference and target gene expression by qRT-PCR. Fold changes in reference genes that fell within (A) and outside (B) the ΔC_T ≤ +/−0.5 limits of suitability. Fold changes in Dusp3 gene expression without (C) and with normalization (D) to 18S, the geometric mean of three reference genes, or β-actin. Asterisks indicates significant differences between treated and untreated samples (p<0.05).

Figure 3. Inaccurate experimental outcome with inappropriate reference gene selection.

Total RNA was isolated over time (0–24 hrs) from density-arrested 3T3-L1 preadipocytes treated with MDI and assessed for reference and target gene expression by qRT-PCR during early stages of differentiation. Fold changes in reference genes that fell within (A) and outside (B) the ΔC_T ≤ +/−0.5 limits of suitability. Fold changes in C/EBPβ gene expression without (C) and with normalization (D) to 18S, cyc, or TfR. Fold changes in cyclin A gene expression without (E) and with normalization (F) to 18S, cyc, or TfR.

Figure 4. Directional shifts in experimental outcome with inappropriate reference gene selection.

Total RNA was isolated over time (0–10 days) from density-arrested 3T3-L1 preadipocytes treated with MDI and assessed for reference and target gene expression by qRT-PCR during complete adipocyte differentiation. Fold changes in reference genes that fell within (A) and outside (B) the ΔC_T ≤ +/−0.5 limits of suitability. Fold changes in PPARγ gene expression without (C) and with normalization to 18S, cyc, or TfR (D), 18S, cyc, or α-tubulin (E), and 18S, cyc, or GAPDH (F). Arrows indicate directional shifts relative to normalization to 18S.