Identification of Appropriate Reference Genes for Human Mesenchymal Cells during Expansion and Differentiation

Rosana Bizon Vieira Carias,Radovan Borojevic,Paola Romina Amable,Pranela Rameshwar,Marcus Vinicius Telles Teixeira,José Mauro Granjeiro

doi:10.1371/journal.pone.0073792

Rosana Bizon Vieira Carias, Radovan Borojevic + Show 4 more

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https://doi.org/10.1371/journal.pone.0073792

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Abstract

BackgroundQuantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately.ResultsIn the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Whartońs Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions.ConclusionMost stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.

Highlights

Real time polymerase chain reaction (PCR) was first described by Higuchi and coworkers in 1992 and the same group reported the first quantitative real time PCR the following year [1,2]
Bestkeeper Results obtained using the BestKeeper software for determining suitable reference genes are shown in Table 2 (AT-mesenchymal stromal cells (MSC)), Table 3 (WJ-MSC), Table 4 (BM-MSC) and Table 5 (DF)
For Wharton’s Jellyderived MSC (WJ-MSC) and dermal fibroblasts (DF), all genes showed an standard deviation (SD) value lower than 1, meaning their expression was stable among all 8 conditions studied and all of them can be used as reference genes

Summary

Introduction

Real time polymerase chain reaction (PCR) was first described by Higuchi and coworkers in 1992 and the same group reported the first quantitative real time PCR the following year [1,2]. A few genes, especially genes involved in basal metabolism such as betaactin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or ribosomal RNA, were believed to be constantly expressed in different cell lines, physiological situations and culture conditions. Such genes were routinely used as reference genes in qPCR, for improving data reliability since they exclude sample to sample variations and RNA quality and sample loading differences. Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. False data can be generated if a reference gene is not chosen adequately

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