Primer Design for Anguilla anguilla

Candidate reference gene products as well as target transcripts and their functions are listed in Table 1. Primers were obtained from previous studies or were designed with Primer Express (Life Technologies) (Table 2), using nucleotide sequences retrieved from the GeneBank database (https://www.ncbi.nlm.nih.gov/genbank/) for Anguilla anguilla or from Next Generation RNA sequencing (454 FLX Titanium) of A. anguilla transcriptome (EeelBase database16 (link)). Primer pair specificities were checked both in silico and empirically by BLAST analysis and using melting profiles. BLAST analyses against A. anguilla nucleotide sequences in the GenBank indicated all primers were specific (data not shown), which was confirmed by melting profiles, electrophoresis, and sequencing of PCR products.