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Europace Advance Access published online on September 27, 2006
Europace, doi:10.1093/europace/eul099
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© The European Society of Cardiology 2006. All rights reserved
Received February 6, 2004
Accepted May 16, 2006

Article

A method for the simultaneous analysis of mRNA levels of multiple cardiac ion channels with a multi-probe RNase protection assay

Yu-ki Iwasaki 1 *, Takeshi Yamashita 2, Akiko Sekiguchi 2, Seiji Hatano 2, Kouichi Sagara 2, Hiroyuki Iinuma 2, Long-Tai Fu 2, Yoshinori Kobayashi 1, Takao Katoh 1, and Teruo Takano 1

1 The First Department of Internal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 1138603, Japan
2 The Cardiovascular Institute, Tokyo, Japan

* To whom correspondence should be addressed.
Yu-ki Iwasaki, E-mail: iwasaki{at}nms.ac.jp


  Abstract

Aims Various pathological conditions can alter cardiac electrophysiological properties not only by physiological responses but also by modifying the gene expression of ion channels (electrical remodelling). To investigate the underlying mechanisms of the latter, electrophysiological alterations would require a simultaneous and comprehensive analysis of the mRNA level of the ion channel genes.

Methods and results We designed 19 cardiac ion channel cDNA templates to analyse the corresponding mRNAs and classified them into three template sets. Those sets were a voltage-dependent K+ channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying K+ channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, {alpha}1C, {beta}2, {alpha}2{delta}2 of cardiac L-type Ca2+ channel and {alpha}1G). These cDNA templates were used to synthesize antisense digoxigenin-labelled RNA probes. An amount of the total RNA of 25 µg was adequate to analyse simultaneously the mRNA levels of the ion channel genes with the use of multi-probe RPA, and these three multi-probe template sets enabled us to evaluate the profile of the spatial and temporal transcripts of the cardiac ion channels.

Conclusion The newly developed ion channel multi-probe RPA templates provide an aid in the comprehensive analysis of the electrical remodelling of the heart.

Keywords: RNase protection assay; Cardiac ion channel; Gene expression; Electrical remodelling; Simultaneous detection.
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