Abstract
Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.
Highlights
Relative quantification using RT-qPCR is a widely used method for transcription profiling
Levels of target gene transcripts are estimated relative to a reference gene shown to be evenly transcribed in the relevant tissues
This assumption has changed into a general understanding that the transcript levels of reference genes may vary considerably [1]
Summary
Cohabitant challenge models represent a natural water borne route of pathogen exposure, and complicate the final analyses as fish sampled at each time-point may be at different stages of infection. It should be emphasized that the number of fish analysed per sampling was low (n = 5), especially considering the cohabitant challenge model applied. This probably resulted in an artificially low intragroup variation. The viral load peaked 3 wpc, only 7 out of 10 animals were positive. At 6 wpc, 9 out of 10 animals were positive
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