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

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.

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

“…Significance was calculated with 2-way ANOVA and Bonferroni post-tests with ***p < 0.001. et al, who showed that HPRT1 was the RG with the highest stability in human bone marrow-derived MSCs and dermal fibroblasts under different expansion and differentiation conditions. 41 PPIA gene encoding for the PPIA is responsible for protein folding processes and was identified as the second and third most stable RG in the combined analysis of 2D and 3D cultured BM-MSCs. In line with our findings, Rienzo et al determined PPIA as an appropriate internal RG in endothelial and osteosarcoma cells for tumor neovascularization studies.…”

Evaluation of the stability of reference genes in bone mesenchymal stem cells from patients with avascular necrosis of the femoral head

“…For example, Studer et al (2012) did so during osteogenesis, adipogenesis, and chondrogenesis of BMSCs and placenta-derived MSCs, concluding that RPL13A is a reliable and stable reference gene. Moreover, in an investigation of reference genes in BMSCs and adipose-and umbilical cord-derived MSCs, Amable et al (2013) established the reliability and stability of B2M and RPL13A. Di et al (2011) analyzed various bone progenitor cell lines under altered gravity conditions and strong magnetic fields, but found no suitable reference genes for such situations.…”

Bioactivity and composition of a preserved connective tissue matrix derived from human placental tissue

“…The control primer sequences were as follows: GAPDH, Forward 5′‐GAG TCA ACG GAT TTG GTC GT‐3′, Reverse 5′‐TTG ATT TTGGAG GGA TCT CG‐3′; RPL13A, Forward 5′‐CCT GGA GGA GAA GAG GAA AGA GA, Reverse 5′‐TTG AGG ACC TCT GTG TAT TTG TCA A‐3′. Previous work identified these reference genes as being stably expressed in ASCs following different culture conditions …”

“…Previous work identified these reference genes as being stably expressed in ASCs following different culture conditions. 26 In vitro suppression of T cell activation Lymphocyte separation medium (Lonza) was used to purify lymphocytes from fresh human blood obtained from healthy donors using density gradient centrifugation. (The use of human tissue samples was approved by the Franciscan University of Steubenville's Institutional Review Board.)…”