Dig rna labeling mix kit

Different developmental stages of S. cephaloptera were sacrificed and pooled for total RNA extraction using RNAqueous™-Micro Total RNA Isolation Kit (Invitrogen GmbH, Karlsruhe, Germany), which was then used for cDNA synthesis using First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche Molecular Biochemicals, Vienna, Austria).
Primers were designed for the identified transcripts using Geneious Prime or Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Reverse primers were synthesized with a T7 promoter sequence (5′-TAATACGACTCACTATAGGG-3′) at the 5’ end. To generate the DNA template for the riboprobe, PCR experiments were performed using these gene-specific primers and Taq 2X Master Mix (New England Biolabs). PCR amplicons were purified with QIAquick PCR Purification Kit (Qiagen Vertriebs GmbH, Vienna, Austria) and were sent to Microsynth Austria for Sanger sequencing to validate their identity. The resulting electropherograms were visually inspected with Geneious Prime. Digoxigenin (DIG)-labeled antisense riboprobes were generated using the T7 polymerase and DIG RNA labeling mix kit (Roche Diagnostics, Mannheim, Germany). Total riboprobe concentrations were estimated using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA). All primer sequences and their annealing temperatures are available in Additional file 2.