Pawlowski V, Révillion F, Hornez L, Peyrat J P
Laboratoire d'Oncologie Moléculaire Humaine, Centre Oscar Lambret, Lille, France.
Cancer Detect Prev. 2000;24(3):212-23.
We developed a real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector System (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time quantification of the polymerase chain reaction products is based on the TaqMan 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer, and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene was used for normalization of c-erbB-2 expression. In human breast cancer cell lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2,600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpressing cells MDA-MB-453 and SK-BR-3. In a series of 100 breast cancer samples, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 350 x 10(-6). A close correlation was observed between this real-time one-step quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) quantified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P < .0001). The current realtime RT-PCR assay is rapid, sensitive, and reproducible and appears particularly suitable to quantify gene expression in large series of samples.
我们开发了一种实时一步法逆转录聚合酶链反应（RT-PCR）方法，用于常规定量检测乳腺癌中c-erbB-2癌基因的表达，该方法使用7700 ABI PRISM序列检测系统（法国库尔塔布夫的珀金埃尔默应用生物系统公司）。聚合酶链反应产物的实时定量基于TaqMan 5'核酸酶测定法。我们确定的最佳实验条件如下：6 mM氯化镁、200 nM荧光探针、200 nM每种引物以及12.5单位莫洛尼鼠白血病病毒逆转录酶。甘油醛-3-磷酸脱氢酶（GAPDH）管家基因用于c-erbB-2表达的标准化。在人乳腺癌细胞系中，c-erbB-2的标准化表达范围为8×10^(-6)至2600×10^(-6)，两个最高值对应于c-erbB-2过表达细胞MDA-MB-453和SK-BR-3。在一系列100个乳腺癌样本中，发现c-erbB-2的标准化表达范围为0.4×10^(-6)至350×10^(-6)。观察到这种实时一步定量RT-PCR方法与半定量常规RT-PCR（N = 22；r = 0.8543；P <.0001）以及通过酶免疫测定（EIA）定量的c-erbB-2蛋白表达（p185）（N = 27；r = 0.71；P <.0001）之间存在密切相关性。当前的实时RT-PCR检测方法快速、灵敏且可重复，似乎特别适合于对大量样本中的基因表达进行定量。
