Fitc rna labeling mix

Total RNA was extracted from 5-day-old female lice with TRI reagent (MRC, Cincinnati, OH, USA) and treated with DNase I (Takara Biotechnology, Shiga, Japan) according to the manufacturer’s protocol. First-strand cDNA was synthesized using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA).
For probe synthesis, LNSP1, LNSP2 and TG fragments were amplified from the female cDNA (primer sequences are shown in Additional file 1: Table S1). For LNSP1 and LNSP2 that show high sequence similarities, respective probe was designed from gene-specific sites in the N terminal domains. The PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI, USA). Each plasmids were digested by ApaI (New England Biolabs, Ipswich, MA, USA) for sense probe (negative control) or NdeI (New England Biolabs) for antisense probe at 37°C for 1 h. The plasmids were checked by electrophoresis to confirm digestion and purified using Monarch^® PCR & DNA Cleanup Kit (New England Biolabs). Sense or antisense LNSP1, LNSP2 and TG probes were generated using T7 or SP6 RNA polymerase (Promega). FITC RNA labeling mix or DIG RNA labeling mix (both from Roche, Mannheim, Germany) were used for LNSP1/LNSP2 probe or TG probes, respectively.

Total RNA was extracted from 5-day-old female lice with TRI reagent (MRC, Cincinnati, OH, USA) and treated with DNase I (Takara Biotechnology, Shiga, Japan) according to the manufacturer's protocol. First-strand cDNA was synthesized using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA).
For probe synthesis, LNSP1, LNSP2 and TG fragments were amplified from the female cDNA (the primer sequences are shown in Additional file 1: Table S1). For LNSP1 and LNSP2, which show high sequence similarities, respective probes were designed from gene-specific sites in the N terminal domains. The PCR products were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). Each plasmid was digested by ApaI (New England Biolabs, Ipswich, MA, USA) for sense probe (negative control) or NdeI (New England Biolabs) for antisense probe at 37 °C for 1 h. The plasmids were checked by electrophoresis to confirm digestion and purified using Monarch® PCR & DNA Cleanup Kit (New England Biolabs). Sense or antisense LNSP1, LNSP2 and TG probes were generated using T7 or SP6 RNA polymerase (Promega). FITC RNA labeling mix or DIG RNA labeling mix (both from Roche, Mannheim, Germany) were used for LNSP1/LNSP2 probe or TG probes, respectively.

The Zswim6 riboprobes were generated as described above. The rat Drd1 and Drd2L riboprobes were kindly provided by K. Kobayashi at Fukushima Medical University, Fukushima, Japan. The riboprobes were synthesized by in vitro transcription with digoxigenin (dig) or fluorescein (FITC) RNA labeling mix (Roche). Briefly, respective linearized template plasmid was mixed with 5× transcription buffer, 0.1 M dithiothreitol (DTT), Dig-labeling mix (Roche), RNasin (Promega), respective RNA polymerase (T3, T7, SP6, Promega), and DEPC-treated H₂O at 37°C for 2 h according to manufacturer’s instruction (Promega). The DNA template was digested with DNase RQ1 at 37°C for 30 min. After stopping polymerase reaction by adding 0.2 M EDTA (pH 8.0) and placed on ice for 5 min, STE buffer (0.1 M NaCl, 10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) and 3 μl 1 M DTT were added. Finally, the probes were further purified with G-50 mini Quick Spin Columns (Roche). The size and quality of probes had been checked by gel electrophoresis before DNase RQ1 treatment and after purification.