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.
Baker
Gregory L
GL
Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616, USA. glbaker@ucdavis.edu
Shultz
Michael A
MA
Fanucchi
Michelle V
MV
Morin
Dexter M
DM
Buckpitt
Alan R
AR
Plopper
Charles G
CG
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