Gene dynamics of core transcription factors for pluripotency in embryonic stem cells

David, Brit G.; Okamoto, Kazuko; Kakizuka, Taishi; Ichimura, Taro; Watanabe, Tomonobu M.; Fujita, Hideaki

doi:10.1016/j.jbiosc.2014.09.011

Cited by 7 publications

(4 citation statements)

References 19 publications

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“…Mouse embryonic stem cell line E14Tg2a was purchased from RIKEN Cell bank, JP (AES0135) and was maintained as previously described [18]. Total RNA was extracted using RNeasy kit (Cat# 74106, Qiagen, Japan) following the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

et al. 2019

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Background The real-time quantitative polymerase chain reaction (qPCR) is routinely used for quantification of nucleic acids and is considered the gold standard in the field of relative nucleic acid measurements. The efficiency of the qPCR reaction is one of the most important parameters in data analysis in qPCR experiments. The Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines recommends the calibration curve as the method of choice for estimation of qPCR efficiency. The precision of this method has been reported to be between SD = 0.007 (three replicates) and SD = 0.022 (no replicates). Results In this article, we present a novel approach to the analysis of qPCR data which has been obtained by running a dilution series. Unlike previously developed methods, our method, Pairwise Efficiency, involves a new formula that describes pairwise relationships between data points on separate amplification curves and thus enables extensive statistics. The comparison of Pairwise Efficiency with the calibration curve by Monte Carlo simulation shows the two-folds improvement in the precision of estimations of efficiency and gene expression ratios on the same dataset. Conclusions The Pairwise Efficiency nearly doubles the precision in qPCR efficiency determinations compared to standard calibration curve method. This paper demonstrates that applications of combinatorial treatment of data provide the improvement of the determination. Electronic supplementary material The online version of this article (10.1186/s12859-019-2911-5) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

et al. 2019

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“…In sequential image acquisition, a time resolution of about 15 min was required to monitor cell division. In a preliminary experiment using 15 min time-lapse imaging of mESCs, we found that the repeated laser illumination strongly compromised the survival of the cells after 48 h 58 . Moreover, during our snapshot experiments (Figs 1 and S1 ), we found that the cells were more sensitive to laser illumination at a certain stage of differentiation, especially during the intermediate stage, from Day-4 to Day-7.…”

Section: Discussionmentioning

confidence: 93%

Single cell analysis reveals a biophysical aspect of collective cell-state transition in embryonic stem cell differentiation

Okamoto¹,

Germond²,

Fujita³

et al. 2018

Sci Rep

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In the stem cell research field, the molecular regulatory network used to define cellular states has been extensively studied, however, the general driving force guiding the collective state dynamics remains to be identified from biophysical aspects. Here we monitored the time-development of the cell-state transition at the single-cell and colony levels, simultaneously, during the early differentiation process in mouse embryonic stem cells. Our quantitative analyses revealed that cellular heterogeneity was a result of spontaneous fluctuation of cellular state and cell-cell cooperativity. We considered that the cell state is like a ball fluctuating on a potential landscape, and found that the cooperativity affects the fluctuation. Importantly, the cooperativity temporarily decreased and increased in the intermediate state of cell differentiation, leading to cell-state transition in unison. This process can be explained using the mathematical equation of flashing-ratchet behaviour, which suggests that a general mechanism is driving the collective decision-making of stem cells.

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“…To make an Oct4-TFP reporter, cDNAs of Oct4 promoter sequence and TFP were amplified with Oct4 reporter used in previous study51 and Golgi-TFP construct, respectively, as a template. The fragments were then cloned into PiggyBac Transposon Vector (System Biosciences) with puromycin resistance marker.…”

Section: Methodsmentioning

confidence: 99%

Protein expression guided chemical profiling of living cells by the simultaneous observation of Raman scattering and anti-Stokes fluorescence emission

Chiu

Ichimura²,

Sekiya

et al. 2017

Sci Rep

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Our current understanding of molecular biology provides a clear picture of how the genome, transcriptome and proteome regulate each other, but how the chemical environment of the cell plays a role in cellular regulation remains much to be studied. Here we show an imaging method using hybrid fluorescence-Raman microscopy that measures the chemical micro-environment associated with protein expression patterns in a living cell. Simultaneous detection of fluorescence and Raman signals, realised by spectrally separating the two modes through the single photon anti-Stokes fluorescence emission of fluorescent proteins, enables the accurate correlation of the chemical fingerprint of a specimen to its physiological state. Subsequent experiments revealed the slight chemical differences that enabled the chemical profiling of mouse embryonic stem cells with and without Oct4 expression. Furthermore, using the fluorescent probe as localisation guide, we successfully analysed the detailed chemical content of cell nucleus and Golgi body. The technique can be further applied to a wide range of biomedical studies for the better understanding of chemical events during biological processes.

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Gene dynamics of core transcription factors for pluripotency in embryonic stem cells

Cited by 7 publications

References 19 publications

Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

Single cell analysis reveals a biophysical aspect of collective cell-state transition in embryonic stem cell differentiation

Protein expression guided chemical profiling of living cells by the simultaneous observation of Raman scattering and anti-Stokes fluorescence emission

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