Northern Blot Analysis of Lung RNA

Northern blots were performed as previously described with some modifications^{1 (link),49 (link)}. Briefly, total RNA extracted from lungs was heat-denatured for 15 min at 75 °C before being electrophoresed on a 1.2% agarose gel containing 7% formaldehyde, alongside an RNA ladder (Life Technologies). Gels were run overnight at 4 °C in MOPS buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA, pH 7.0). RNA was transferred by capillary transfer to a nylon membrane (Hybond N+, Amersham Biosciences) overnight after which the membrane was crosslinked with UV light (120 mJ/cm²) and hybridised with [α³²P]-dCTP and [α³²P]-dATP labelled probes diluted in hybridisation buffer (Ambion Ultrahyb) overnight at 42 °C. Primer sequences used to generate the probes are listed in Table 1.