Validation of reference genes for transcription profiling in the salmon louse, Lepeophtheirus salmonis, by quantitative real-time PCR.

The salmon louse Lepeophtheirus salmonis, a marine ectoparasitic copepod that feeds on salmonids, cause huge financial losses in the aquaculture industry through fish mortality, reduced growth and therapeutic costs. Detailed knowledge about the salmon louse life cycle, at the functional molecular level, is of fundamental importance to evaluate alternative therapeutic or prophylactic strategies. In quantitative real-time PCR, a powerful technique in biological studies of differentially expressed genes, the transcription level of a regulated gene can be measured relative to an unregulated reference "housekeeping" gene. In the present study we validate candidate reference genes for transcription profiling throughout the life cycle of the salmon louse. Our results show that the structural ribosomal protein S20 (RPS20) and the translation elongation factor 1alpha (eEF1alpha) are valid as reference genes showing less than two fold variation in transcript levels. The frequently used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), however, is up to six fold regulated during the L. salmonis life cycle. Furthermore our results indicated that 18S RNA, although constitutively expressed, is not a convenient reference gene for relative quantification of most transcripts.