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

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

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 highvolume samples such as spheroids. Thismethod achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

Original languageEnglish
Pages (from-to)275-282
Number of pages8
JournalAnalytical and Bioanalytical Chemistry
Volume406
Issue number1
DOIs
Publication statusPublished - 2014 Jan

Keywords

  • Angiogenesis
  • Differentiation
  • RT-PCR
  • Real-time PCR
  • Single-cell analysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

Fingerprint Dive into the research topics of 'Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe'. Together they form a unique fingerprint.

  • Cite this