Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction
Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction
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DOI：
10.1016/j.ab.2005.03.034
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发表时间：
2005-07-01
影响因子：
2.9
通讯作者：
Gillies, RJ
中科院分区：
文献类型：
--
作者：
Morse, DL;Carroll, D;Gillies, RJ
实时逆转录酶聚合酶链式反应被认为是一种高度灵敏且特异的 mRNA 表达定量方法。 SYBR green I 染料简化了实验设计，但需要特定的对照来保持高特异性。由于灵敏度的提高，对于不太敏感的方法的标准化可能可接受的标准已被证明在细胞系、组织、增殖状态、治疗和发育条件以及癌症进展程度之间存在很大差异。很明显，当该方法应用于任何新的实验模型时，确定合适的标准化标准是使用该方法的必要条件。我们评估了一些常用标准对一组转移潜力增加的人类乳腺癌细胞系中 mRNA 标准化的适用性，并确定 I SS rRNA 和 β-肌动蛋白 (A CTB) mRNA 均适合此目的，但各自都有一些局限性。 18S rRNA 在细胞系之间变化较小，但具有较高程度的随机变异性，而 A CTB mRNA 在细胞系之间变化较大，但具有较低程度的随机变异性。 (c) 2005 Elsevier Inc. 保留所有权利。
Real-time reverse transcriptase polymerase chain reaction is recognized as a highly sensitive and specific method for quantification of mRNA expression. SYBR green I dye simplifies the experimental design but introduces the need for specific controls to maintain high specificity. Due to this increased sensitivity, standards that may have been acceptable for normalization of less sensitive methods have been shown to vary considerably among cell lines, tissues, proliferative states, treatments, and developmental conditions and by degree of cancer progression. It has become evident that determination of suitable normalization standards is a requirement for the use of this method as it is applied toward any new experimental model. We have assessed the suitability of a number of commonly used standards for the normalization of mRNAs among a set of human breast cancer cell lines of increasing metastatic potential and have determined that I SS rRNA and beta-actin (A CTB) mRNA are both suitable for this purpose, with each having some limitations. 18S rRNA varies less among the cell lines but has a higher degree of random variability, while A CTB mRNA varies more among cell lines but has a lower degree of random variation. (c) 2005 Elsevier Inc. All rights reserved.