Selection of suitable reference genes for reverse transcription-quantitative polymerase chain reaction analysis of neuronal cells differentiated from bone mesenchymal stem cells

Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a technique widely used for the quantification of mRNA transcription, It is well recognized that the reference genes used in RT-qPCR require appropriate validation to ensure that gene expression is unaffected by experimental conditions. The differentiation of bone mesenchymal stem cells (BMSCs) into neurons is important in the treatment of nerve injury. In gene expression analysis of the differentiation of BMSCs into neuronal cells by, the commonly used reference genes for RNA analysis are often selected without any preliminary evaluation of their suitability. The present study aimed to evaluate the mRNA expression levels of 11 putative reference genes, including ACTB, ARBP, B2M, CYCA, GAPDH, GUSB, HPRT, PPIA, RPL13A, TBP and PGK1, in order to select the most suitable reference genes for RT-qPCR of the differentiation of neuronal cells by BMSCs. The mRNA expression levels of the 11 putative reference genes were examined using RT-qPCR in rat BMSCs differentiated into neuronal cells. Normal BMSCs and three types of rat BMSCs, which were chemically induced to differentiate into neurons using neurotrophic cytokines and co-culture with retinal cells. The geNorm, NormFinder and BestKeeper software programs were used to select the most suitable reference genes. The results of the analyses using the three software programs demonstrated that RPL13A was the most stable among all the groups, while ACTB was the least stable. The combination of CYCA and PPIA reference genes contributed the most to increasing stability. The suitability of selected reference genes requires previous pre-selection in every investigation. Based on the three software programs, RPL13A, and the combination of CYCA and PPIA were identified as the most suitable reference genes for RT-qPCR in neuronal cells differentiated from BMSCs.

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

“…These differences resulted from the heterogeneous gene expression profile of the tumor type, the experimental conditions, and the stability of the reference genes, being in agreement with the literature (Supplementary Table 1) [21,22,28,29]. In addition, the aforementioned variability can also be attributed to the different algorithms applied by the GeNorm and NormFinder [23,[30][31][32][33]. However, for the single HKG analysis, the first three ranked genes by GeNorm and NormFinder were the same ones, implying no substantial differences in their stability.…”

Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

“…In general, internal reference genes, such as 18S rRNA, GAPDH, and ACTB, are chosen for relative quantification analysis between clinical samples. However, increasing evidence has suggested that the expression levels of these commonly used internal reference genes are variable in distinct tissues, cell lines, between treatments of the same cell line (Ali et al, 2015; Ma et al, 2015; Yang et al, 2014; Yu et al, 2015), as well as across cell types (He et al, 2015; Li et al, 2015a; Li et al, 2015b). Thus, with the advancement of precise medicine, it is of high importance to evaluate and validate internal reference genes for the target gene expression profile studies among different cell types and tissues.…”

Identifying suitable reference genes for developing and injured mouse CNS tissues

“…The complex spatial and temporal alterations after injury could further complicate interpretation of changes in gene expression (Gauthier et al, ; Yu and He, ). Therefore, reference genes that are frequently used for other experiments may be unsuitable for studies of neural development or regeneration (He et al, ). Although attempts have been made to search for reference genes in several other tissues, few studies have focused on reference genes in the developing brain and spinal cord from prenatal, postnatal to aging periods or under injury conditions.…”