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ToxSci Advance Access published online on November 4, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfh002
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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Received May 16, 2003; accepted September 22, 2003
© 2003 Society of Toxicology

Respiratory Toxicology

Assessing Gene Expression in Lung Subcompartments Utilizing in Situ RNA Preservation

Gregory L. Baker 1*, Michael A. Shultz 2, Michelle V. Fanucchi 1, Dexter M. Morin 3, Alan R. Buckpitt 3, and Charles G. Plopper 1

1 Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616
2 Global Research, Amersham Biosciences, Sunnyvale, CA 94086; Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616
3 Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616

* To whom correspondence should be addressed. E-mail: glbaker{at}ucdavis.edu.


  Abstract

The mechanisms of toxicant mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity significant differences and similarities in gene expression are observed throughout lung subcompartments. RNA based technologies such as real time reverse transcription polymerase chain reaction (real time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high quality RNA could be preserved in situ, over a period of time, to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Two hours after 1-nitronaphthalene treatment, rat lungs were inflated with and stored in RNA preservation solution and stored at 4°C for 7 days. RNA was isolated from lung subcompartments isolated by microdissection. After 7 days of storage, the RNA was intact, of high quality and could be used for real time RT-PCR to examine heterogeneous gene expression in lung subcompartments. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high quality RNA that may be used with molecular RNA based technologies that will significantly accelerate our understanding of pulmonary injury and repair mechanisms.

Key Words: Lung, Heterogeneity, Microdissection, RNA, Preservation, Real Time PCR, Airways, Parenchyma .


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