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Issue 11, 2011
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Ribonuclease protection assay on microchip electrophoresis

Yuka Yamaguchi,ab   Shouki Yatsushiro,*a   Shohei Yamamura,a   Hiroko Abe,a   Kaori Abe,a   Masahiro Watanabe,a   Kazuaki Kajimoto,c   Yasuo Shinohara,b   Yoshinobu Babaad  and  Masatoshi Kataokaa  
Author affiliations

* Corresponding authors

a Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Japan
E-mail: yatsushiro.s@aist.go.jp
Fax: +81-87-869-3553
Tel: +81-87-869-3597

b Graduate School of Pharmaceutical Sciences, University of Tokushima, Shoumachi 1-78, Tokushima, Japan

c Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo, Hokkaido, Japan

d Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan

Abstract

We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.

Graphical abstract: Ribonuclease protection assay on microchip electrophoresis

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Article information

https://doi.org/10.1039/C0AN01044H
Submitted
30 Dec 2010
Accepted
11 Mar 2011
First published
21 Apr 2011
Analyst, 2011,136, 2247-2251
Article type
Paper
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Ribonuclease protection assay on microchip electrophoresis

Y. Yamaguchi, S. Yatsushiro, S. Yamamura, H. Abe, K. Abe, M. Watanabe, K. Kajimoto, Y. Shinohara, Y. Baba and M. Kataoka, Analyst, 2011, 136, 2247
DOI: 10.1039/C0AN01044H

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