Amersham hyperfilm mp autoradiography film

RNA from infected cells or virus suspension was extracted using TRIzol reagent (Invitrogen). RNA samples were prepared using NorthernMax formaldehyde loading dye (Ambion) and 1 μl of ethidium bromide and then heated to 65°C for 20 min. Samples were then separated on a 1.2% low-electroendosmosis (LE) agarose (Lonza) gel containing 1× morpholinepropanesulfonic acid (MOPS) running buffer (Ambion) and 6.7% formaldehyde. RNA was transferred onto nitrocellulose membrane overnight and then cross-linked by UV irradiation (UVP), and the membrane was blocked for 1 h at 68°C in ULTRAhyb ultrasensitive hybridization buffer (Ambion). RNA probes complementary to positive-strand RNA were then synthesized using a MAXIscript SP6 or T7 in vitro transcription kit (Ambion) and labeled with ³²P. After removal of unincorporated nucleotides using Illustra MicroSpin S200 HR columns, probes were hybridized to membranes overnight at 68°C. Membranes were then washed three times with washing buffer (0.1× SSC–0.1% SDS in autoclaved water; 1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 68°C for 20 min and then imaged using Amersham Hyperfilm MP autoradiography film (GE Healthcare).

The total RNAs purified from wild-type fruiting body and mycelium were separated with 15% acrylamide/8 M urea gel and transfer to the Hybond-N+ nitrocellulose membrane (GE healthcare, Piscataway, NJ). The specific isotope probes were label with γ-[³²P] ATP (3000 Ci/mmol, PerkinElmer, Waltham, MA) using T4 polynucleotide kinase (NEB, Ipswich, MA). Then the membrane exposed to the Amersham hyperfilm MP autoradiography film (GE healthcare, Piscataway, NJ).