Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference.
Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference.
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DOI：
10.1093/clinchem/45.2.297
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发表时间：
1999-02
影响因子：
9.3
通讯作者：
K. Kreuzer;U. Lass;O. Landt;A. Nitsche;J. Laser;H. Ellerbrok;G. Pauli;D. Huhn;C. Schmidt
中科院分区：
文献类型：
--
作者：
K. Kreuzer;U. Lass;O. Landt;A. Nitsche;J. Laser;H. Ellerbrok;G. Pauli;D. Huhn;C. Schmidt
使用常见的逆转录（RT）-PCR参考靶序列可通过扩增污染DNA或加工假基因产生假阳性结果。此外，仅定性RT-PCR不能区分高质量和低质量的cDNA制备物，这对于解释低丰度转录本可能是至关重要的。我们已经开发了一种高灵敏度的定量RT-PCR β-actin，使用TaqManTM化学。通过该技术，我们能够定量检测每100 ng cDNA中的10个β-肌动蛋白分子，而无需共扩增假基因或基因组DNA。因此，所提出的方法可能是有利的定量RT-PCR结果的解释。PCR分析可能难以解释，除非平行扩增参考靶序列。该对照反应对于评价所研究样品中是否存在足量的可液化物质是必要的。因此，只有当参考基因的扩增显示阳性结果时，阴性PCR才能被定义为阴性PCR。扩增参考在RT-PCR中尤其重要，因为RNA可以在cDNA合成之前或期间迅速降解。作为RT-PCR的参考序列，优选所谓的“管家”基因，因为它们由所有细胞类型组成型表达。β-肌动蛋白是参考共扩增的有吸引力的候选物，因为它仅表现出微小的个体内动力学变化，并且主要不受任何人类疾病的影响（1）。然而，β-肌动蛋白和其他常用参考文献（如甘油醛-3-磷酸脱氢酶）的主要问题是，加工的假基因可以通过最初限制于cDNA的引物共扩增。这个问题很难解决，因为mRNA和相应的假基因之间存在>90%的序列同源性（2）;因此，假基因通常不能在凝胶电泳中区分。此外，只要不能安全地排除基因组DNA污染，DNA可以与cDNA一起共扩增。因此，积极分析…
The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results. PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis. As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …