Mileham K A, Northover B J, Winter A W, Hundal S P, Brammar W J, Conley E C
University of Leicester/Medical Research Council, Centre for Mechanisms of Human Toxicity, UK.
Mol Cell Probes. 1994 Apr;8(2):161-76. doi: 10.1006/mcpr.1994.1022.
We have measured the expression levels of a range of distinct ion channel genes in the apex/ventricle region and sino-atrial node (SAN) sub-regions of heart under conditions in which conventional Northern hybridization or ribonuclease protection methods were too insensitive or non-quantitative. The abundance of six potassium channel mRNAs was determined in relation to a single synthetic competitor RNA template which was co-reverse transcribed and PCR-amplified. By these methods we have shown that coronary artery ligation procedures which induce anoxia and ischaemic scarring in the apical region reduce amplifiable message abundance in a time-dependent, but non-specific manner. There was no evidence for any selective reduction of individual mRNA levels during this process. Despite a high reproducibility of titration endpoints, competitive RNA template amplification assays did not provide a simple marker for ischaemic damage, since it was not possible to control for tissue sample heterogeneity. We have also applied these competitive nucleic acid titration techniques to demonstrate expression of cAMP- and cGMP-gated ion channel sequences in small pieces of tissue derived from the SAN sub-region of rabbit heart. Although the ion channels encoded by these sequences are obligately coupled to intracellular signalling agonists commonly found in cardiac cells, they have not been described in functional terms within SAN or any other cardiac subregion. For rapid determination of cDNA molecular numbers, we have devised single-gene, DNA template controls to measure absolute abundance of a cAMP-gated cDNA derived from heart tissue. Competitive titration procedures therefore provide an important technique for probing gene induction and/or repression accompanying pharmacological or surgical interventions, or in progression of disease states. For rare cDNAs, they can estimate the representativeness of a given preparation prior to library construction, screening and retrieval of clones, while eliminating 'false positive' or 'false negative' signals.
我们已经测量了一系列不同离子通道基因在心脏的心尖/心室区域和窦房结（SAN）子区域中的表达水平，这些条件下传统的Northern杂交或核糖核酸酶保护方法过于不灵敏或无法定量。相对于单一的合成竞争RNA模板，测定了六种钾通道mRNA的丰度，该模板经共反转录和PCR扩增。通过这些方法，我们表明，在顶端区域诱导缺氧和缺血性瘢痕形成的冠状动脉结扎程序以时间依赖性但非特异性的方式降低了可扩增信息的丰度。在此过程中，没有证据表明单个mRNA水平有任何选择性降低。尽管滴定终点具有高度可重复性，但竞争性RNA模板扩增试验并未提供缺血性损伤的简单标志物，因为无法控制组织样本的异质性。我们还应用了这些竞争性核酸滴定技术来证明兔心脏SAN子区域来源的小块组织中cAMP和cGMP门控离子通道序列的表达。尽管这些序列编码的离子通道与心脏细胞中常见的细胞内信号激动剂紧密偶联，但它们在SAN或任何其他心脏子区域内尚未从功能角度进行描述。为了快速测定cDNA分子数量，我们设计了单基因DNA模板对照来测量源自心脏组织的cAMP门控cDNA的绝对丰度。因此，竞争性滴定程序为探究伴随药理或手术干预或疾病状态进展的基因诱导和/或抑制提供了一项重要技术。对于罕见的cDNA，它们可以在文库构建、克隆筛选和检索之前估计给定制备物的代表性，同时消除“假阳性”或“假阴性”信号。
