Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation

Abstract

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance

“…For quantitative PCR, the increase of fluorescence of the SYBR green dye was measured using Platinum® SYBR-Green qPCR SuperMix-UDG, 10 ng cDNA, 1.2 µM primer mix, and a CFX Connect™ Real-Time PCR Detection System (Bio-Rad) as advised by the manufacturer. Data were normalized to beta-actin which served as recommended housekeeping gene [49]. For primer sequences see Supplementary Table S2.…”

Multimodal Label-free Monitoring of Adipogenic Stem Cell Differentiation using Endogenous Optical Biomarkers

“…To further confirm the adipogenic lineage, we investigated the gene expression level of adiponectin [53], leptin [54], and PPARg [55], which were found to be associated with the promotion of adipogenic differentiation. The mRNA fold change of these adipogenic genes were calculated and normalized to cyclophilin B gene by 2 -ΔΔCT method [56,57]. All the gene expression levels increased for hASCs cultured for 9 days in adipogenic media compared to 6 days culture.…”

Dual Regulatory Role of Polyamines in Adipogenesis

“…Real time PCR analysis was performed using SYBR Green I (Kapa Biosystems) as a fluorescent dye, according to the manufacturer's instructions. All experiments were carried out in triplicates, and results were normalized to HMBS RNA, as recommended by Zhang et al (26). Real time PCR primers were selected from the Harvard Primer Bank (Primer Bank IDs are as follows: CHOP-31982415a1; HMBS-30794512a1).…”