A method for isolation of rat renal microvessels and mRNA localization.
The objective of this study was to develop a technique to identify and dissect segments of the rat renal microcirculation and to apply reverse transcription (RT) to specific mRNAs with subsequent amplification of the cDNA by polymerase chain reaction (PCR) to evaluate gene expression. To circumvent the difficulty associated with visualizing specific microvessels, we intrarenally infused blue latex microparticles, 1-5 microns in diameter, with subsequent identification and microdissection of specific segments of the renal microvasculature under stereomicroscopy. To demonstrate its utility, we assessed expression of mRNAs encoding fibronectin and renin. As expected, mRNA encoding fibronectin was localized along the renal microcirculation, and mRNA encoding renin was primarily present in afferent arterioles and interlobular arteries. Identity of the amplified cDNA fragments was verified by sequencing. This perfusion-microdissection technique coupled to RT-PCR should be useful in the evaluation of gene expression along the renal microvasculature. It may also allow bridging of the gap between analysis of gene expression of rare mRNA species by in situ hybridization and physiology of the renal microcirculation.
Pelayo
J C
JC
Department of Pediatrics, University of California, Los Angeles, School of Medicine 90024.
Mobilia
M A
MA
Tjio
S
S
Singh
R
R
Nakamoto
J M
JM
Van Dop
C
C
D000818:Animals; D001808:Blood Vessels / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D006652:Histological Techniques; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008833:Microcirculation; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012079:Renal Circulation; D012083:Renin / Q000235:genetics; D014018:Tissue Distribution
D005353:Fibronectins; D012333:RNA, Messenger; D012083:Renin