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Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe

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Abstract

In this study, we introduce the double-barrel carbon probe (DBCP)—a simple, affordable microring electrode—which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

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Acknowledgments

This research was partially supported by the Cabinet Office, Government of Japan, through its “Funding Program for Next Generation World-Leading Researchers” (to H.S) and by a Grant-in-Aid for Advanced Measurement and Analysis from the Japan Science and Technology Agency (JST). Y.N acknowledges the support obtained from a research fellowship of the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai, 980-8579, Japan

    Yuji Nashimoto, Ryosuke Takano, Kosuke Miyashita, Shukuyo Yamada, Kosuke Ino, Hitoshi Shiku & Tomokazu Matsue

  2. WPI-Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai, 980-8577, Japan

    Yasufumi Takahashi & Tomokazu Matsue

Authors
  1. Yuji Nashimoto
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  2. Yasufumi Takahashi
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  3. Ryosuke Takano
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  4. Kosuke Miyashita
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  5. Shukuyo Yamada
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  6. Kosuke Ino
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  7. Hitoshi Shiku
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  8. Tomokazu Matsue
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Corresponding authors

Correspondence to Hitoshi Shiku or Tomokazu Matsue.

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Nashimoto, Y., Takahashi, Y., Takano, R. et al. Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe. Anal Bioanal Chem 406, 275–282 (2014). https://doi.org/10.1007/s00216-013-7430-z

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  • DOI: https://doi.org/10.1007/s00216-013-7430-z

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