Detection and quantitation of EP3 prostaglandin E2 receptor mRNA along mouse nephron segments by RT-PCR

Abstract

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.