EOSINOPHILIC LUNG INFLAMMATION IN PARTICULATE-INDUCED LUNG INJURY - TECHNICAL CONSIDERATION IN ISOLATING RNA FOR GENE-EXPRESSION STUDIES

Particulate and other pollutant exposures are associated with lung inj ury and inflammation. The purpose of this study was to develop an appr oach by which intact RNA could be obtained from inflamed lung tissue f rom particulate-exposed animals in order to correlate injury with spec ific gene expression. Male Sprague Dawley (SD) and Fischer-344 (F-344) rats were intratracheally instilled with saline or residual oil fly a sh (ROFA) instillation, lungs were either lavaged or used for RNA isol ation. ROFA exposure produced an increase in bronchoalveolar lavage fl uid (BALF) neutrophils in both SD and F-344 rats. A time-dependent inc rease in eosinophils occurred only in SD rats but not in F-344 rats. E xtraction if inflamed pulmonary tissue having a high influx of eosinop hils for RNA using the conventional acid guanidinium thiocyanate pheno l-chloroform (AGPC) procedure failed to provide undergraded RNA suitab le 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 extra cted from lung tissues having eosinophil influx. Evidently, the conven tional AGPC procedure failed to completely remove ribonuclease activit y 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 step s for extracting undergraded RNA from nuclease-rich inflamed lung tiss ue. The aqueous layer resulting from mixing homogenate and chloroform is extracted a second time using an equal volume of AGPC buffer follow ed by addition of chloroform and centrifugation. This simple aqueous p hase is then treated as described in the conventional RNA extraction p rotocol. This simple aqueous phase is then treated as described in the conventional RNA extraction protocol. This sample and convenient modi fication does not require multiple precipitations of RNA and yields un dergraded RNA from inflamed lung tissue with a slightly higher A(260)/ A(280) ratio without affecting overall RNA recovery. The results indic ate that undergraded RNA could not be isolated using the routine ROFA exposure. The degraded RNA preparations were unsuitable for gene expre ssion studies. However, undergraded RNA can be isolated from these tis sues by modifying the original AGPC RNA extraction procedure, which is suitable for gene expression analysis using northern blot and RT-PCR techniques.