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Mol Neurobiol. 2019 Mar 29. doi: 10.1007/s12035-019-1538-x. [Epub ahead of print]

Reference Gene Validation via RT-qPCR for Human iPSC-Derived Neural Stem Cells and Neural Progenitors.

Author information

1
Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland.
2
Department of Neurochemistry, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland. jlenart@imdik.pan.pl.
3
Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, 5a Pawinskiego Str., 02-106, Warsaw, Poland.
4
Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland. buzanska@imdik.pan.pl.

Abstract

Correct selection of the reference gene(s) is the most important step in gene expression analysis. The aims of this study were to identify and evaluate the panel of possible reference genes in neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP) obtained from human-induced pluripotent stem cells (hiPSC). The stability of expression of genes commonly used as the reference in cells during neural differentiation is variable and does not meet the criteria for reference genes. In the present work, we evaluated the stability of expression of 16 candidate reference genes using the four most popular algorithms: the ΔCt method, BestKeeper, geNorm and NormFinder. All data were analysed using the online tool RefFinder to obtain a comprehensive ranking. Our results indicate that NormFinder is the best tool for reference gene selection in early stages of hiPSC neural differentiation. None of the 16 tested genes is suitable as reference gene for all three stages of development. We recommend using different genes (panel of genes) to normalise RT-qPCR data for each of the neural differentiation stages.

KEYWORDS:

Gene expression reference panel; Neural Progenitor; Neural Stem Cells; Relative gene expression; human induced Pluripotent Stem Cell (hiPSC); quantitative real-time Polymerase Chain Reaction (RT-qPCR)

PMID:
30927132
DOI:
10.1007/s12035-019-1538-x

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