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

Europace (2006) 8, 1011–1015 doi:10.1093/europace/eul099 A method for the simultaneous analysis of mRNA levels of multiple cardiac ion channels with a multi-probe RNase protection assay Yu-ki Iwasaki1*, Takeshi Yamashita2, Akiko Sekiguchi2, Seiji Hatano2, Kouichi Sagara2, Hiroyuki Iinuma2, Long-Tai Fu2, Yoshinori Kobayashi1, Takao Katoh1, and Teruo Takano1 1 2 The First Department of Internal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 1138603, Japan and The Cardiovascular Institute, Tokyo, Japan Received 6 February 2004; accepted after revision 16 May 2006; online publish-ahead-of-print 27 September 2006 KEYWORDS RNase protection assay; Cardiac ion channel; Gene expression; Electrical remodelling; Simultaneous detection 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 Kþ channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying Kþ channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, a1C, b2, a2d2 of cardiac L-type Ca2þ channel and a1G). These cDNA templates were used to synthesize antisense digoxigenin-labelled RNA probes. An amount of the total RNA of 25 mg 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. Introduction Many pathological conditions including atrial fibrillation,1 cardiac hypertrophy, and heart failure2 have been reported to alter the cardiac electrophysiological properties that may lead to arrhythmogenesis, i.e. ‘electrical remodelling’.3 This remodelling is mainly believed to result from the alteration of ionic currents through modification of gene expression. As cardiac ion channels are diverse, in order to discuss the substrates for arrhythmogenesis, it is necessary to obtain comprehensive information about the gene expression of many of the ion channels. The RNase protection assay (RPA) is known as a highly sensitive method for the quantification of specific mRNAs.4 Recently, RPA has been used for the simultaneous detection of multiple target genes using multi-probes.5 In fact, the simultaneous quantification of many chemokines has been * Corresponding author. Tel: þ81 3 3822 2131 ext. 6743; fax: þ81 3 5685 0987. E-mail address: reported to be useful. In cardiac electrophysiology, RPA has been used to analyse a single target gene.6,7 One of the reasons is that the mRNA of cardiac ion channels has a high homologous sequence to each other, which makes the simultaneous analysis difficult. However, a method for the simultaneous analysis of multiple genes with limited samples would provide great help also in cardiac electrophysiology. We developed three multi-probe RPA template sets that allowed for the simultaneous and comprehensive analysis of the mRNA levels of cardiac voltage-dependent potassium channels, inwardly rectifying potassium channels, and sodium and calcium channels. Methods Animal and total RNA preparation Sprague-Dawley rats aged 10 weeks were used in the present study. The hearts were removed, and the atria and ventricles were excised and quickly frozen in liquid nitrogen and stored at 2808C. To avoid contamination of the tissues, only right and left appendages were & The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: 1012 Y.-K. Iwasaki et al. used as the atrial samples. The total RNA was extracted using the acid guanidinium isothiocyanate method.8 The total RNA concentration was measured with a spectrophotometer at a wave length of 260 nm. Table 1 Sequence, probe length, and accession number of RNA probes Kv series Erg KvLQT1 Kv4.3 Kv4.2 Kv2.1 Kv1.5 Kv1.4 Kv1.2 Kir series Kir3.1 Kir6.2 SUR2 Kir2.2 Kir2.1 Kir3.4 Cin series b2 a2d2 a1C a1G SCN5A Internal control Cardiac troponin T Cyclophilin Sequence Probe length (nt) Accession number 40–464 1–390 811–1150 310–599 1952–2191 2648–2838 1877–2026 1887–2006 435 390 340 290 240 190 150 120 U75210 U92655 L48619 M59980 X16476 M27158 M32867 J04731 1000–1550 2062–2153 4472–4832 1201–1466 252–450 1911–2060 550 452 361 266 199 150 NM031610 D86039 D83598 X78461 AF021137 U01071 1693–2084 291–630 1239–1525 3842–4085 1561–1760 392 340 287 245 200 NM053851 AF042792 AF394940 AF027984 NM013125 921–980 60 105–204 100 M26052, J04995 M19533 Multi-probe templates We classified 19 cardiac ion channel genes into three groups according to the characteristic of each gene. They were a voltagedependent Kþ channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying Kþ channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, a1C, b2, a2d2 of cardiac L-type Ca2þ channel and a1G), referred to as the Kv series, Kir series, and Cin series, respectively. All the cardiac ion channel genes were known to be expressed in the rat heart. Cardiac troponin T was used as internal control. Alignment confirmation For an optimal setting of the multi-probe templates, we carefully selected the sub-cloning site for specific RNA probes. First, for genes already known to have splicing variants, i.e. Kv4.3 and Kir3.1,9,10 variant sites were excluded to avoid making a ladder band in the RPA. Secondly, the maximum highly homologous nucleotide sequences in the channel genes were detected and excluded. To evaluate the homologous alignment within the ion channel genes in each group, cluster analysis of those genes was preformed using an alignment programme (clustal X).11 Thirdly, only protected fragments that had at least a 10% difference in length were selected to differentiate well between each protected fragment. Thereafter, the nucleotide sequence of the primers was determined for the subcloning site of the RNA probes (Table 1 and 2). To prepare the cDNA templates of the 19 cardiac ion channel genes, a reverse transcription–polymerase chain reaction (RT–PCR, 30 cycles at 948C for 30 s, at 60–628C for 30 s, and at 728C for 90 s, with an Access RT–PCR system, Promega, WI, USA), using the total RNA isolated from the rat atria as a template, was perform (...truncated)