Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development.

Antisense RNA probes were synthesized by in vitro transcription incubating 1 μg linearized plasmid, 2 μL 100 mM dithiothreitol (DTT), 1.3 μL NTP mixture (15.4 mM each ATP, CTP, GTP, and 10 mM UTP), 0.7 μL 10 mM digoxigenin-UTP or fluorescein-UTP (Roche Applied Science, Indianapolis, IN, USA), and 0.5 μL RNAguardTM (Amersham Biosciences, Piscataway, NJ, USA), 2 μL 10× transcription buffer, 1 μL RNA polymerase (20 U/μL; Roche Applied Science) in a final volume of 20 μL for 2–4 h at 37°C. After the addition of 1 μL DNase I (Roche Applied Science), the reaction was incubated for additional 15–30 min. RNA probes were cleaned using the RNeasy® mini kit (Qiagen, Valencia, CA, USA) and eluted in 50 μL double-distilled water, of which 2 μL were controlled by gel electrophoresis. The remainder was mixed with 75 μL hybridization mixture and stored at -20°C. Hybridization Mixture