GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells

Abstract

BackgroundQuantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR.Materials and methodsAdipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon.ResultsThe expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively.ConclusionThis study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

TNF‐α Upregulates IKKε Expression via the Lin28B/let‐7a Pathway to Induce Catecholamine Resistance in Adipocytes

“…Total protein was harvested from cultured cells with ice-chilled radioimmunoprecipitation assay lysis buffer (ThermoFisher Scientific There are several limitations we have to acknowledge. In 2015, Nazari et al reported that GAPDH and β-actin were not appropriate for normalization of mRNA in adipocytes or adipose tissue (20). We conducted some experiments using both β-actin and tubulin as reference genes, and the results are consistent with previous data in which tubulin was used as the reference gene.…”

Identification of Optimal Reference Genes for Expression Analysis in Radish (Raphanus sativus L.) and Its Relatives Based on Expression Stability

“…Medrano found that GAPDH was one of the best genes in combination for embryonic samples (Medrano et al, 2017 ). Our results were consistent with these previous studies suggesting that GAPDH can also be universally used as a reference gene in radish and their relatives, although several reports in animal and plant systems have suggested that GAPDH has some limitations as an internal control gene (Sirover, 1999 ; Nazari et al, 2015 ). The reference gene UP2 , encoding an uncharacterized conserved protein, is related to cell structure.…”

Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue

“…It is generally known that GAPDH and β-actin are the two most commonly-used reference genes. However, some researchers have identified that GAPDH and β-actin showed a significant variation in different cells, tissues and experimental conditions [24,25,26]. Furthermore, Catalan and his colleagues showed that GAPDH is a less appropriate reference gene in human omental and subcutaneous adipose tissue from obesity and type 2 diabetes patients, because of the variational expression [27].…”