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Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming
Yulia Panina1,2, Arno Germond1, Shinji Masui3
1RIKEN Center for Biosystems Dynamics Research (BDR), 6-2-3 Furuedai, Suita, Osaka, 565-0874, Japan.
Scientific Reports
|June 9, 2018
Summary
This study analyzed housekeeping gene stability during induced pluripotent stem cell (iPS) reprogramming. We identified the most stable genes (Atp5f1, Pgk1, Gapdh) for accurate gene expression analysis in iPS cells.
Area of Science:
- Stem cell biology
- Molecular biology
- Genetics
Background:
- Induced pluripotent stem cell (iPS) reprogramming involves significant cellular changes.
- Accurate gene expression analysis during iPS reprogramming requires validated reference genes for methods like RT-qPCR.
- Lack of studies evaluating reference gene stability during iPS reprogramming.
Purpose of the Study:
- To assess the stability of 12 housekeeping genes during a 20-day iPS reprogramming process in murine cells.
- To identify reliable reference genes for accurate gene expression studies in iPS reprogramming.
- To provide crucial data for researchers investigating cell-state and pluripotency during iPS reprogramming.
Main Methods:
- Analysis of 12 housekeeping genes using RT-qPCR data over 20 days of iPS reprogramming.
- Application of five different statistical algorithms to evaluate gene stability.
- Validation using a proof-of-point qPCR experiment with pluripotent markers (Nanog, Rex1, Oct4).
Main Results:
- Significant variations in housekeeping gene stability were observed during iPS reprogramming.
- Atp5f1, Pgk1, and Gapdh were identified as the most stable housekeeping genes.
- Rps18, Hprt, Tbp, and Actb demonstrated the least stability.
Conclusions:
- Housekeeping gene stability is highly variable during iPS reprogramming, necessitating careful selection of reference genes.
- Atp5f1, Pgk1, and Gapdh are recommended as stable reference genes for RT-qPCR in iPS reprogramming studies.
- This research provides essential guidance for accurate gene expression profiling in pluripotency and cell-state investigations.
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