Format

Send to

Choose Destination
PLoS One. 2010 Dec 13;5(12):e15208. doi: 10.1371/journal.pone.0015208.

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

Author information

1
Department of Nutrition, The University of North Carolina at Greensboro, Greensboro, North Carolina, United States of America.

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.

PMID:
21179435
PMCID:
PMC3001470
DOI:
10.1371/journal.pone.0015208
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center