Taniguchi S, Watanabe T, Nakao A, Seki G, Uwatoko S, Kurokawa K
First Department of Internal Medicine, University of Tokyo, Japan.
Am J Physiol. 1994 May;266(5 Pt 1):C1453-8. doi: 10.1152/ajpcell.1994.266.5.C1453.
mRNA of the EP3 prostaglandin E2 (PGE2) receptor was detected and quantitated in microdissected mouse nephron segments by a modified protocol of reverse transcription-polymerase chain reaction (RT-PCR). At the step of RT, a point mutation was introduced in cDNA, which made a new restriction site for the Mbo I enzyme. PCR was performed using a set of primers on the same exon, and genomic DNA was coamplified with cDNA by these primers. PCR products were treated with Mbo I, and signals from genomic DNA and mRNA were separately detected on gel electrophoresis. The relative amount of mRNA per cell was expressed as a ratio of amount of product from mRNA to that from genomic DNA. EP3 PGE2 receptor mRNA expression was abundant in thick ascending limb of Henle, present to a lesser extent in distal convoluted tubules and collecting ducts, and undetectable in glomeruli, proximal tubules, and descending thin limb. The results support the notion that PGE2 modulates water and solute transport through the EP3 receptor in specific structures of the kidney.
通过改良的逆转录-聚合酶链反应（RT-PCR）方案，在显微切割的小鼠肾单位节段中检测并定量了前列腺素E2（PGE2）受体EP3的mRNA。在逆转录步骤中，cDNA中引入了一个点突变，这为Mbo I酶创造了一个新的限制性酶切位点。使用同一外显子上的一组引物进行PCR，这些引物同时扩增基因组DNA和cDNA。PCR产物用Mbo I处理，在凝胶电泳上分别检测基因组DNA和mRNA的信号。每个细胞中mRNA的相对量表示为mRNA产物量与基因组DNA产物量的比值。EP3 PGE2受体mRNA在亨氏袢升支粗段中大量表达，在远曲小管和集合管中表达程度较低，在肾小球、近端小管和降支细段中未检测到。这些结果支持了PGE2通过EP3受体在肾脏特定结构中调节水和溶质转运的观点。
