RT-qPCR analyses on the osteogenic differentiation from human iPS cells: An investigation of reference genes

Abstract

Reverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem (iPS) cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days.RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein (TBP) in the first place, followed by transferrin receptor (TFRC), ribosomal protein large P0 (RPLP0), and finally, beta-2-microglobulin (B2M), which was revealed as the least stable.Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.Quantitative real-time PCR (qPCR) was developed in the 1990s 1 and is now widely used in various fields as a tool for the accurate and easy detection as well as quantification of target nucleic acid samples. In particular, reverse transcription quantitative PCR (RT-qPCR) is regarded as an indispensable experimental technique for analysis of gene expression by quantifying RNA.RT-qPCR is used to measure the amplification of an exponentially amplified region and quantify its initial template cDNA, that is, mRNA. Accurate assessments require the correction of inter sample variations, particularly of factors such as the total amount and quality of RNA, reverse transcription efficiency, and PCR efficiency. To perform this, the internal control genes are selected as reference genes, which are quantified along with the target gene.Standardization is performed by calculating their expression ratios. The "housekeeping genes", those with constant expression levels in the tissues or cells to be analyzed, are generally used as internal control genes; however, their expression levels may vary each tissue and each cell. Fluctuations have been reported depending on the conditions such as the stages of development 2 . The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE), a set of guidelines compiled to ensure the reproducibility of qPCR, stipulate that reference genes suited for each cell, tissue, and experimental design be selected from numerous candidates so as to ensure reliability of normalisation 3 . Therefore it is evident that, the selection of suitable reference genes, along with the provision of data demonstrating their expression stability, is becoming an indispensable part of quantitative analysis of gene expression using RT-qPCR.To date, research has mainly focused on the osteogenic potential of mesenchymal stem cells (MSCs) 4,5 .However, since Takahashi and Yamanaka 6 reported about human induced p...

Mesenchymal stem cell culture in aligned porous hydroxyapatite scaffolds using a multiwell plate bioreactor for bone tissue engineering

“…In the current study, first, the self‐renewal and differential potential of the commercially obtained hWJ‐MSCs was confirmed by culturing the cells on PLM and ODM for 3 weeks. As presented in Figure 2, the results presented in this report match with previous reports, which showed similar changes in morphology and differential alizarin red staining for cells cultured in PLM versus ODM 26,27 . Subsequently, first‐hand information on hWJ‐MSC infiltration into the bioceramic cages was obtained by observing the cell‐laden scaffolds under a microscope as well as by performing Alamar blue assay.…”

“…As presented in Figure 2, the results presented in this report match with previous reports, which showed similar changes in morphology and differential alizarin red staining for cells cultured in PLM versus ODM. 26,27 Subsequently, first-hand information on hWJ-MSC infiltration into the bioceramic cages was obtained by observing the cell-laden scaffolds under a microscope as well as by performing Alamar blue assay. As presented in Figure 3, our results indicated relatively higher cell infiltration and proliferation on aligned porous scaffolds compared to random porous ones.…”