Edelstein R A, Carr M C, Caesar R, Young M, Atala A, Freeman M R
Urology Research Laboratory, Children's Hospital, Boston, MA 02115.
DNA Cell Biol. 1994 Mar;13(3):265-73. doi: 10.1089/dna.1994.13.265.
A competitive polymerase chain reaction (PCR) method for analysis of androgen receptor (AR) mRNA expression is described. The technique involves the use of an in vitro-transcribed RNA (cRNA) corresponding to a region of the AR mRNA transcript as a competitor in reverse transcription and PCR (RT-PCR) using total cellular RNA. The competitor RNA contains a site-directed mutation that produces a restriction fragment length polymorphism after RT-PCR and endonuclease digestion. We demonstrate that incorporation of the competitor RNA into RT-PCR reactions allows rapid semiquantitative determination of relative AR mRNA levels without the necessity of following PCR product formation kinetically; reaction products are assessed at the conclusion of the reaction sequence and without the use of radioactive probes or other specialized detection systems. We have used competitive PCR to demonstrate low levels of AR mRNA in an androgen-unresponsive human prostate cell line (PC3). In addition, we have also used this method to confirm that genital fibroblasts obtained from a subject with penoscrotal hypospadias (a non-intersex masculinization defect) that exhibit low levels of high-affinity androgen binding also exhibit abnormally low AR mRNA levels. These last results suggest that some non-intersex malformations of the urogenital tract are associated with abnormalities in the expression of the androgen receptor.
本文描述了一种用于分析雄激素受体（AR）mRNA表达的竞争性聚合酶链反应（PCR）方法。该技术涉及使用与AR mRNA转录本区域相对应的体外转录RNA（cRNA）作为竞争者，与总细胞RNA一起用于逆转录和PCR（RT-PCR）。竞争者RNA包含一个位点定向突变，该突变在RT-PCR和核酸内切酶消化后产生限制性片段长度多态性。我们证明，将竞争者RNA掺入RT-PCR反应中可以快速半定量测定相对AR mRNA水平，而无需动态跟踪PCR产物的形成；在反应序列结束时评估反应产物，且无需使用放射性探针或其他专门的检测系统。我们使用竞争性PCR证明了雄激素无反应性人前列腺癌细胞系（PC3）中AR mRNA水平较低。此外，我们还使用该方法证实，从患有阴茎阴囊型尿道下裂（一种非两性畸形的男性化缺陷）的受试者获得的生殖成纤维细胞，其高亲和力雄激素结合水平较低，AR mRNA水平也异常低。这些最新结果表明，一些泌尿生殖道的非两性畸形与雄激素受体表达异常有关。
