Dig or fluorescein rna labeling mix

DIG- and fluorescein-labeled RNA probes were prepared by in vitro transcription with RNA polymerases and plasmid vectors containing target transcript sequences. One μg of linearized plasmid DNAs was used as a template, and RNA probes were synthesized with SP6, T7 or T3 RNA polymerase (Roche) and DIG or Fluorescein RNA Labeling Mix (Roche) for 2–3 h at 37 °C. After precipitation with ethanol and lithium chloride, the RNA probes were dissolved in DDW. After determining the concentrations, the RNA probes were diluted (50 ng/μl) with probe dilution buffer (50% formamide, 5X SSC, 0.1% Tween-20) containing torula RNA (5 mg/ml) and were stored at − 20 °C.
In this study, we prepared 8 DIG-labeled antisense and sense RNA probes for the full lengths of mouse Pou5f1/Oct4 and zebrafish cyclin B1 gene transcripts and for the parts of zebrafish dazl and mouse Dazl gene transcripts (Additional file 1: Table S2). In addition, we prepared 6 fluorescein-labeled antisense and sense RNA probes for the full lengths of zebrafish mos, zebrafish cyclin B1, and mouse Cyclin B1 gene transcripts (Additional file 1: Table S2). Two DIG-labeled and 2 fluorescein-labeled antisense and sense RNA probes for the full length of lncRNA-HSVIII were also prepared (Additional file 1: Table S2). Sequences of the all transcripts used for making RNA probes were shown in Additional file 1: Table S3.

To isolate the H. austinesis slit and robo gene, previously published sequences, we used BLAST implemented in the whole draft-genome reference (http://genome.jgi.doe.gov/Helro1/Helro1.home.html). Three candidate genes (protein id: Hau-slit 103882; Hau-robo1 189275; Hau-robo2 188972) were screened. In the A. lata transcriptome data, one slit and two robo transcripts were found. The slit and robo specific primers were designed to amplify the fragments (For detailed primer sequences, see Additional file 1: Table S1). These amplified fragments were cloned into pGEM T vector (Promega, Madison, WI, USA). RNAprobes labeled with digoxigenin or fluorescein were made using the MEGAscript kit (Ambion, Austin, TX, USA) and DIG or Fluorescein RNA Labeling Mix (Roche, Basel, Switzerland), according to the manufacturer's instructions. The synthesized RNA probes were applied to each sample at a final concentration of 2 ng/μl.