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.
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.
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