RNA Isolation and Northern Blot Analysis

Total RNA from phenotypic and non-phenotypic larvae at 120 hpf was isolated using phenol:chloroform extraction. 2 μg of total RNA per lane was subjected to electrophoresis on a 0.8% agarose gel (with the addition of 7% formaldehyde) in 1xMOPS/1% formaldehyde buffer. RNA was then blotted to a Hybond-Nylon membrane (Amersham) and subsequently visualized using methylene blue staining. The detection of rRNA precursors was performed using the High Prime DNA Labeling and Detection Starter Kit II (Roche) kit, according to manufacturer’s protocol. The DNA probes targeted 5’ETS, ITS1, and ITS2 rRNA [61 (link)]. The signal was detected using an X-ray film (Kodak).