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- Title
RT-qPCR analyses on the osteogenic differentiation from human iPS cells: an investigation of reference genes.
- Authors
Okamura, Kensuke; Inagaki, Yusuke; Matsui, Takeshi K.; Matsubayashi, Masaya; Komeda, Tomoya; Ogawa, Munehiro; Mori, Eiichiro; Tanaka, Yasuhito
- 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 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 in the first place, followed by transferrin receptor, ribosomal protein large P0, and finally, beta-2-microglobulin, 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.
- Subjects
POLYMERASE chain reaction; REVERSE transcriptase polymerase chain reaction; COMPUTER algorithms; CARRIER proteins; OSTEOBLASTS
- Publication
Scientific Reports, 2020, Vol 10, Issue 1, p1
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-020-68752-2