title: Eosinophilic lung inflammation in particulate-induced lung injury: technical consideration in isolating RNA for gene expression studies.
abstract: Particulate and other pollutant exposures are associated with lung injury and inflammation. The purpose of this study was to develop an approach by which intact RNA could be obtained from inflamed lung tissue from particulate-exposed animals in order to correlate injury with specific gene expression. Male Sprague Dawley (SD) and Fischer-344 (F-344) rats were intratracheally instilled with saline or residual oil fly ash (ROFA) particles, 8.3 mg/kg body weight in saline. At various time points following ROFA instillation, lungs were either lavaged or used for RNA isolation. ROFA exposure produced an increase in bronchoalveolar lavage fluid (BALF) neutrophils in both SD and F-344 rats. A time-dependent increase in eosinophils occurred only in SD rats but not in F-344 rats. Extraction of inflamed pulmonary tissue having a high influx of eosinophils for RNA using the conventional acid guanidinium thiocyanate phenol-chloroform (AGPC) procedure failed to provide undegraded RNA suitable for RT-PCR and Northern blot analysis of beta-actin mRNA expression. Mixing intact total RNA from saline control rat lungs with degraded RNA samples from inflamed lung yielded a gel profile of degraded RNA, indicating the presence of ribonuclease-like activity in the RNA extracted from lung tissues having eosinophil influx. Evidently, the conventional AGPC procedure failed to completely remove ribonuclease activity associated with ROFA-induced pulmonary eosinophil influx. This study reports a single-step modification to the AGPC extraction method that does not require additional reagents or additional precipitation steps for extracting undegraded RNA from nuclease-rich inflamed lung tissue. The aqueous layer resulting from mixing homogenate and chloroform is extracted a second time using an equal volume of AGPC buffer followed by addition of chloroform and centrifugation. The second aqueous phase is then treated as described in the conventional RNA extraction protocol. This simple and convenient modification does not require multiple precipitations of RNA and yields undegraded RNA from inflamed lung tissue with a slightly higher A260/A280 ratio without affecting overall RNA recovery. The results indicate that undegraded RNA could not be isolated using the routine AGPC-based isolation technique from lung tissue containing eosinophils following ROFA exposure. The degraded RNA preparations were unsuitable for gene expression studies. However, undegraded RNA can be isolated from these tissues by modifying the original AGPC RNA extraction procedure, which is suitable for gene expression analysis using northern blot and RT-PCR techniques.
authors.affiliation: Pulmonary Toxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
authors.forename: U P
authors.initials: UP
authors.lastname: Kodavanti
authors.forename: R H
authors.initials: RH
authors.lastname: Jaskot
authors.forename: J
authors.initials: J
authors.lastname: Bonner
authors.forename: A
authors.initials: A
authors.lastname: Badgett
authors.forename: K L
authors.initials: KL
authors.lastname: Dreher
mesh_terms: D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002244:Carbon / Q000008:administration & dosage / Q000633:toxicity*; D002452:Cell Count; D060729:Coal Ash; D004804:Eosinophils; D005786:Gene Expression Regulation; D007220:Industrial Waste / Q000009:adverse effects; D007249:Inflammation / Q000139:chemically induced / Q000473:pathology*; D007442:Intubation, Intratracheal; D008168:Lung / Q000737:chemistry / Q000473:pathology; D008171:Lung Diseases / Q000139:chemically induced / Q000473:pathology*; D008297:Male; D052638:Particulate Matter; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases / Q000032:analysis
chemical_list: D060729:Coal Ash; D007220:Industrial Waste; D052638:Particulate Matter; D012313:RNA; D002244:Carbon; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases