*2.8. Riboprobe Synthesis*

mRNA probes to identify NfuNT-6 mRNA were synthetized by in vitro transcription (IVT) using MAXIscript™ SP6/T7 in vitro transcription kit (Invitrogen by Thermo Fisher Scientific–Catalogue number AM1312, Carlsbad, CA, USA) and following the manufacturer's instructions. 1 μg of DNA template was transcribed to RNA in 20 μL volume reaction, using NfuNT6 primer associated with the T7 promoter sequence (left 5- -TGGTCCTGCTCTTCCTGATC-3- ; T7 right 5- -GGTAATACGACTCACTATAGG\_GTGTGTTTGAAGCTGCTCGA-3- ) and a DIG RNA Labeling Mix, 10× conc (Roche, Basel, Switzerland) containing digoxigenin labeled uracil. After IVT reaction, product was briefly centrifuged and incubated at 37 ◦C for 1 h. Then, 1 μL of turbo DNase 1 was added, sample was mixed well and incubated for 15 min at 37 ◦C. 1 μL of EDTA 0.5 M was added to stop the reaction. Reaction product was analyzed by gel electrophoresis and quantized.
