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


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, alpha1C, beta2, alpha2delta2 of cardiac L-type Ca(2+) channel and alpha1G). These cDNA templates were used to synthesize antisense digoxigenin-labelled RNA probes. An amount of the total RNA of 25 microg 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.


CONCLUSIONS
The newly developed ion channel multi-probe RPA templates provide an aid in the comprehensive analysis of the electrical remodelling of the heart.
Iwasaki
Yu-Ki
YK
The First Department of Internal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 1138603, Japan. iwasaki@nms.ac.jp
Yamashita
Takeshi
T
Sekiguchi
Akiko
A
Hatano
Seiji
S
Sagara
Kouichi
K
Iinuma
Hiroyuki
H
Fu
Long-Tai
LT
Kobayashi
Yoshinori
Y
Katoh
Takao
T
Takano
Teruo
T
D000818:Animals; D001681:Biological Assay / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D015336:Molecular Probe Techniques; D009206:Myocardium / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases
D007473:Ion Channels; D012333:RNA, Messenger; D012260:Ribonucleases