Dig rna labelling mix

One µg of total RNAs in a denaturing buffer were run on a 1% agarose gel, transferred and chemically crosslinked to Hybond-N (GE Healthcare). 28 S RNA was detected with methylene blue. After washing, membranes were hybridized with probes in DIG Easy Hyb Granules (Sigma Aldrich) at 65 °C. The region encoding Cdkn1c in the plasmid^{42 (link)} was transcribed with T3 promoter (antisense probe) and T7 promoter (sense probe), labeled with DIG RNA Labelling Mix (Roche) and used as probes. The images were acquired by ImageQuant LAS4000 (GE Healthcare) and quantified using ImageQuant TL (GE Healthcare).

For in situ hybridization experiments, tissue preparation, sectioning and transcript detection were performed as described42 (link). A MC probe was prepared using cDNA as template (393-pb fragment of Solyc05g056620.1.1) and excluding the MADS-box domain. Antisense transcripts were synthesized using the DIG RNA labelling mix (Roche Applied Science). As negative control, sense RNA probes were hybridized with the same sections and no signals were observed under the hybridization and detection conditions used.

Pre-hybridization was carried out by incubating the sections in 0.2M HCl, neutralizing them and then treating them with 1mg/ml proteinase-K (Roche Applied Science). Samples were then dehydrated in an aqueous ethanol dilution series and hybridized with sense and anti-sense digoxigenin (DIG)-labelled RNA probes, corresponding to DNA fragments (200–300bp) derived from the 3′-non coding regions of the BdMAN2, BdMAN4 and BdMAN6 genes (Supplementary Table S3), synthesized with the DIG RNA labelling mix according to the manufacturer’s specifications (Roche Applied Science). Probes were hybridized at 52ºC overnight followed by two washes in 2× SSC (150mM NaCl, 15mM Na₃-citrate) and 50% formamide for 90min at the same temperature. Incubation with the alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Applied Science) and colour detection was carried out according to the manufacturer’s instructions (Ferrandiz et al., 2000 (link)). Sections were dried and examined on a Zeiss Axiophot Microscope (Carl Zeiss, Oberkochen, Germany), and images were captured and processed with the Leica Application Suite 2.8.1 build software (Leica).

Gene-specific primers (Additional File 6: Table S5) were designed using Primer3 [177 (link)] implemented in Geneious v.8 (Biomatters) against predicted gene model sequences. Parameters were set to produce optimal product sizes of 1–1.5 Kb and to anneal at least 50 bp internally from the ends to increase their efficiency in cases where template cDNAs were not full length. Transcripts were amplified by PCR from cDNAs synthesised from total RNA purified from either Whole Adult or pooled, larval worms. Products were cloned using a StrataClone PCR cloning kit (Agilent Technologies) according to the manufacturer’s instructions, except that all volumes were halved. Transformed plaques were picked and added to 500 μl water, heated to 80 °C to liberate the plasmids, and the resulting mixture used as template for further amplification by PCR using M13 primers. Resulting products (consisting of the gene insert flanked by T3/T7 reverse transcriptase promoter regions and M13 priming sites) were cleaned, quantified and used as templates for probe synthesis. Digoxigenin (DIG)-labelled riboprobes were synthesised from both the sense (+) and anti-sense (−) strands using T3 and T7 reverse transcriptases and DIG-RNA labelling mix (Roche).

Shh probes were transcribed from a plasmid described by Echelard et al. (1993) (link), by means of in vitro transcription with the incorporation of digoxigenin-ddUTP, using a premixed DIG RNA labelling mix (Roche). In situ hybridisation was performed with a conventional protocol. The antibody utilised was an anti-DIG antibody coupled with alkaline phosphatase (Roche); the chromogenic substrate used was BM Purple ready-to-use NBT/BCIP (Roche). Samples were examined with Leica M205 stereomicroscope. Images were taken with the Leica Application Suite 4.1 software package.

cDNA was synthesized with MMLV reverse transcriptase (Evrogen) from total RNA extracted from larvae with TRIzol reagent (Thermo Scientific). Primers specific for the 543 bp fragment were designed based on full-length transcript from the H. dujardini transcriptome (F: 5′-CAGTTCAACGACGAGATTTG-3′; R: 5′-CCTGAAAGATGTGCCTTTG-3′). The fragment was cloned in pAL-2T vector (Evrogen). Inserts were verified by Sanger sequencing and used for probe synthesis. Antisense digoxigenin-labelled RNA probe was made by in vitro transcription with DIG RNA labelling mix (Roche) and an appropriate RNA polymerase (Thermo Scientific). WMISH of the larvae was performed as described [20 (link)]. Alkaline phosphatase-labelled probe was visualized with the NBT/BCIP colorimetric substrate system (Roche).
To confirm the existence of different transcripts, cloning was performed as follows. cDNA was synthesized with random primers from total RNA. Primers were designed in such a way as to amplify CDS and at least part of both 5′ and 3′ UTRs (electronic supplementary material, file S2). PCR was carried out with theTersus Plus PCR kit (Evrogen). PCR products were cloned in pAL2-T plasmid (Evrogen) and subjected to Sanger sequencing.