PPIA, HPRT1, and YWHAZ Genes Are Suitable for Normalization of mRNA Expression in Long-Term Expanded Human Mesenchymal Stem Cells

Abstract

Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

“…Conversely, in the same study, BestKeeper identifies GUSB as the most stable reference gene, demonstrating that stability can differ among methods and an overall ranking is required [36]. Various studies have also suggested that YWHAZ may be a suitable reference gene in monolayer cultures [26,37], however we observe that YWHAZ is not an appropriate choice of reference gene in 2D and 3D HA-based osteogenic cultures, which supports the findings of Brinkdorf et al [36] that YWHAZ is not suited for 3D culture. A further point concerns the commonly used reference gene 18S, which we consider as an unsuitable choice as a reference gene, despite its apparent stability in 3D cultures.…”

TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

“…Considering the most common RGs in MSCs from other species, ten types of RGs were selected and designed using the NCBI Primer Designing Tool ( http://www.ncbi.nlm.-nih.gov/tools/primer-blast/ ), resulting in a PCR amplicon with 80 to 130 base pairs at an annealing temperature of 60°C ( Table 1 ) [ 1 , 7 – 9 ]. The qPCR was conducted using a Rotor Gene Q qPCR machine (Qiagen, Germany) with Rotor-Gene 2× SYBR Green mix (Qiagen, Germany), including 0.1 μg cDNA per reaction and 0.5 mM forward and reverse primers of RGs.…”

“…To validate the PCR efficiency of each RG in bBMMSCs, a standard curve of each primer of RG was generated from the Ct values using a four-fold serial dilution of cDNA from bBMMSCs under the aforementioned qPCR condition. The values related to PCR efficiency (E) and correlation (R 2 ) were obtained using Excel (Microsoft, Redmond, WA, USA) as described in a previous report [ 7 ].…”

“…In this context, quantitative real-time polymerase chain reaction (qPCR), which has the advantages of convenience, sensitivity, reproducibility, and reliability, has been most commonly used to verify the potential of MSCs and determine the change in the mRNA expression of genes of interest (GOIs) [ 1 , 2 ]. During qPCR, the GOI is normalized against a reference gene (RG), also known as a housekeeping gene, as an internal control for its relative quantification; this step corrects sample-to-sample variations in the context of different experimental conditions, sample quality, operators, and laboratories [ 1 , 2 , 7 , 8 ]. Therefore, RGs should be stably expressed in various samples and not be affected by various experimental conditions; in principle, RGs play a pivotal role in the vital functions of cell survival and maintenance [ 7 , 9 ].…”

“…It is known that the expression of RGs is also variable depending on the experimental conditions and cell types [ 10 ]. In particular, the normalization of GOIs against inadequate or unstable RGs may result in false or contradictory conclusions [ 1 , 7 , 11 ]. Therefore, validation of RGs for their stability under each experimental condition is an extremely important and prerequisite step for obtaining reliable results during qPCR assay [ 2 , 7 , 8 ].…”