Extraction and Quantification of RNA from Zebrafish (Danio rerio) Olfactory Tissue

Abstract

Nucleic acids extraction from tissues is the first step toward sequencing and quantifying gene expression. This work is part of a larger project that seeks to understand changes in gene expression in zebrafish olfactory tissues in response to treatment with 11-keto-testosterone (11-KT), a steroid hormone. Previous work has shown that exposure of juvenile zebrafish to (11-KT) results in sexual behaviors that mimic those of mature zebrafish. Our interest lies in characterizing the expression of a novel family of olfactory receptors (oras) that have been shown in teleost fish, and have sequence similarity to pheromone receptors. In this study, we extracted RNA from treated and control zebrafish samples olfactory epithelia and olfactory bulbs using an organic extraction method. The RNA samples will be sent to an outside company for sequencing and bioinformatics analyses, which typically require a minimum of 20 ug of RNA. Simultaneously, we extracted RNA from fruit fly (Drosophila melanogaster) larvae and compared yields. It should be noted that other methods exist for isolation of nucleic acid (i.e. columnbased methods), however, the use of organic solvents to isolate RNA is preferred in this case, as column-based methods typically lose about 20% of RNA. Our proximate purpose is to characterize and quantify expression of specific olfactory receptors (oras), however, we are also seeking to create a transcriptome library of gene (messenger RNA) expression. Our results show that the yield of RNA from juvenile zebrafish olfactory epithelia and olfactory bulbs is very low and so, we may need to pool RNA samples.