Assessing gene expression in lung subcompartments utilizing in situ RNA preservation.
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.
Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616, USA. glbaker@ucdavis.edu
Gregory L
GL
Baker
Michael A
MA
Shultz
Michelle V
MV
Fanucchi
Dexter M
DM
Morin
Alan R
AR
Buckpitt
Charles G
CG
Plopper
D000818:Animals; D016477:Artifacts; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D005404:Fixatives; D015870:Gene Expression / Q000187:drug effects; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D042282:Microdissection; D009281:Naphthalenes / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014021:Tissue Preservation / Q000379:methods*
D017931:DNA Primers; D015342:DNA Probes; D005404:Fixatives; D009281:Naphthalenes; D012333:RNA, Messenger; C016614:1-nitronaphthalene