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Assessing gene expression in lung subcompartments utilizing in situ RNA preservation.
- Source :
-
Toxicological sciences : an official journal of the Society of Toxicology [Toxicol Sci] 2004 Jan; Vol. 77 (1), pp. 135-41. Date of Electronic Publication: 2003 Nov 04. - Publication Year :
- 2004
-
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 degrees C for 7 days. RNA was isolated from the 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 the 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.
- Subjects :
- Animals
Artifacts
DNA Primers chemistry
DNA Probes chemistry
Electrophoresis, Agar Gel
Fixatives
Lung drug effects
Lung pathology
Male
Microdissection
Naphthalenes toxicity
RNA, Messenger genetics
RNA, Messenger metabolism
Rats
Rats, Sprague-Dawley
Gene Expression drug effects
Lung metabolism
RNA, Messenger isolation & purification
Tissue Preservation methods
Subjects
Details
- Language :
- English
- ISSN :
- 1096-6080
- Volume :
- 77
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Toxicological sciences : an official journal of the Society of Toxicology
- Publication Type :
- Academic Journal
- Accession number :
- 14600286
- Full Text :
- https://doi.org/10.1093/toxsci/kfh002