Primers for kcnq1-5 and controls were designed based on zebrafish DNA sequences found in publically available databases such as the NCBI (GenBank), and Ensembl.
The primers used for amplification are based on zebrafish gapdh cDNA sequence (GenBank accession number BC095386): gapdh: 5′-GATACACGGAGCACCAGGTT-3′, 5′-CGTTGAGAGCAATACCAGCA-3′.
Amplification of a partial sequence of the dax1 gene of S. pyrenaicus and S. alburnoides was performed using primers based on zebrafish dax1 (ENSDART00000020212) (Table 1).
Based on zebrafish dataset, a total of 24,281 Ensembl zebrafish proteins were matched by catfish contigs, corresponding to 20,014 unique genes (Table 5).
To investigate the role of these genes in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression and disease correlation.
The zebrafish 14K oligo microarray comprising 14,067 zebrafish oligonucleotides was designed and synthesized by MWG Genomic Company (Germany) based on 1800 zebrafish gene sequences from the NCBI and a database of 12,768 putative ORFs using NCBI zebrafish EST sequence information [50].
We narrowed down a list of 69 GWAS candidate genes in 42 loci to 14 zebrafish genes based on available zebrafish orthologs, zebrafish and/or liver gene expression pattern, and known disease correlation.
Based on the zebrafish fabp10 sequence [ 24], Her et al., [ 22] cloned its promoter and by functional analysis, identified a 435 bp regulatory element that is sufficient to modulate the liver regional expression in transgenic zebrafish.
This cDNA was used as a template for PCR with sequence specific primers designed based on the zebrafish bmp16 transcript sequence found in Ensembl (ID: ENSDART00000098426).
Based on the zebrafish paralogous NUCB2 sequences, exon 2 for NUCB2A is 20 base pairs larger than exon 2 in NUCB2B.
