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.