Osada T, Hirata S, Hirai M, Hagihara K, Kato J
Department of Obstetrics and Gynecology, Yamanashi Medical School, Japan.
Endocr J. 1993 Aug;40(4):439-46. doi: 10.1507/endocrj.40.439.
In order to examine the existence and level of androgen receptor messenger ribonucleic acid (ARmRNA) in the rat brain, reverse transcription-polymerase chain reaction (RT-PCR)-Southern blot analysis was carried out. Total RNA extracted from each tissue was reverse transcribed, followed by PCR with two oligonucleotide primers specific for a part (458 bp) of the androgen binding domain of the rat ARcDNA. It was confirmed by direct nucleotide sequencing that the amplified fragment corresponded to part of the rat ARcDNA. To detect and quantify the amplified fragments, a Southern blot analysis was carried out. The levels of amplified fragments were calculated from the standard curve obtained from graded diluted adrenal total RNAs. In the present study, it was revealed that the RT-PCR-Southern blot analysis possessed a high-degree of sensitivity and allowed the quantitative estimation of mRNA. With this method, amplified fragments were obtained from all five brain regions examined. The results indicate that ARmRNA is widely distributed in the whole brain. Moreover, since the ARmRNA level roughly paralleled the AR protein level, it seems that the AR protein level in the brain may be primarily regulated by the ARmRNA level.
为了检测大鼠脑中雄激素受体信使核糖核酸（ARmRNA）的存在及水平，进行了逆转录-聚合酶链反应（RT-PCR）-Southern印迹分析。从每个组织中提取的总RNA进行逆转录，然后用两个针对大鼠ARcDNA雄激素结合域一部分（458 bp）的寡核苷酸引物进行PCR。通过直接核苷酸测序证实扩增片段对应于大鼠ARcDNA的一部分。为了检测和定量扩增片段，进行了Southern印迹分析。扩增片段的水平根据从分级稀释的肾上腺总RNA获得的标准曲线计算得出。在本研究中，发现RT-PCR-Southern印迹分析具有高度敏感性，并能对mRNA进行定量估计。用这种方法，从所检测的所有五个脑区都获得了扩增片段。结果表明ARmRNA广泛分布于整个大脑。此外，由于ARmRNA水平大致与AR蛋白水平平行，看来脑中的AR蛋白水平可能主要受ARmRNA水平的调节。
