Targeted AMP amplification in Anura. Anura

Antimicrobial peptides (AMPs) are endogenous peptide antibiotics present in all organisms as part of innate immunity. Amphibians are a particularly rich source of these peptides, which act as multifunctional effector molecules often combining antimicrobial activities with signaling or immunomodulatory functions. Traditionally, identification of frog AMPs required to collect and to sacrifice several specimens. Frogs housed in tanks would be usually stimulated to obtain sufficient quantities of skin secretion to allow the isolation of AMPs from active fractions. We propose an efficient and less invasive alternative approach to selectively amplify AMP-coding transcripts from small amounts of tissue samples. This protocol includes RNA extraction and cDNA synthesis, followed by PCR amplification with a forward degenerate primer, designed on the highly conserved signal peptide region of the AMP precursor, and a reverse primer targeting the mRNA poly-A tail. We tailored primer design to enable the specific amplification of sequences from Ranidae, Hylidae and Bombinatoridae based on sequence data available in public databases. Size-selected amplicons were processed by high-throughput ion semiconductor sequencing and the sequencing output was assembled into contigs. The virtual protein translation of these sequences allowed the identification of more than one hundred full-length mature peptides, including a number of novel AMPs.