Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction.
We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.
Moriyama
T
T
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Murphy
H R
HR
Martin
B M
BM
Garcia-Perez
A
A
D000449:Aldehyde Reductase / Q000235:genetics*; D000818:Animals; D005069:Evaluation Studies as Topic; D007678:Kidney Glomerulus / Q000032:analysis; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000032:analysis; D007687:Kidney Tubules, Proximal / Q000032:analysis; D009399:Nephrons / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics*; D014018:Tissue Distribution; D014158:Transcription, Genetic
D012333:RNA, Messenger; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase