Comparison of regional variability in lung-specific gene expression using a novel method for RNA isolation from lung subcompartments of rats and mice.
The lung is composed of a complex assemblage of more than 40 different cell types. Therefore, investigative techniques that rely on samples derived from whole lung homogenates, whether for biochemical measurements of metabolism or the analysis of gene expression, are inherently insensitive to cell type or region-specific differences. Microdissection has previously been successful for defining region-specific metabolic activity in the lung. Tissues obtained by this technique exhibit good viability and permit reproducible enzyme activity measurements. In this paper, a technique for isolating RNA from lung subcompartments obtained by microdissection is described. The method is straight forward and results in high quality RNA that can be used to quantify specific mRNAs in microscopically selected lung subcompartments by complementary DNA or RNA hybridization techniques. This technique provides a significant increase in sensitivity over techniques based on whole lung homogenates because RNA contributed by relevant lung subcompartments is enriched. The high sensitivity of the method makes it feasible to compare differences in mRNA expression 1) within different regions of the lung in the same animal, 2) in the same region in different animals and between different species, and 3) between susceptible and nonsusceptible sites in conditions of focal lung injury.
Royce
F H
FH
Department of Pediatrics, University of California, Davis, CA 95817, USA.
Van Winkle
L S
LS
Yin
J
J
Plopper
C G
CG
D000818:Animals; D001059:Apoproteins / Q000235:genetics*; D015152:Blotting, Northern; D001980:Bronchi / Q000378:metabolism; D018076:DNA, Complementary; D004210:Dissection; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D006801:Humans; D008168:Lung / Q000033:anatomy & histology / Q000378:metabolism* / Q000601:surgery; D008297:Male; D051379:Mice; D011506:Proteins / Q000235:genetics*; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014598:Uteroglobin
D001059:Apoproteins; D018076:DNA, Complementary; D011506:Proteins; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; C556389:SCGB1A1 protein, human; C556390:Scgb1a1 protein, mouse; C046616:pulmonary surfactant apoprotein; D012313:RNA; D014598:Uteroglobin