Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals

Next Generation Sequencing technologies (NGS) are rapidly invading many evolutionary and ecological fields, such as phylogenomics, molecular evolution, population genomics and molecular ecology. Among the potential targets of NGS is transcriptome sequencing, a fast and relatively cheap way to generate massive amounts of coding sequence data, offering promising perspectives for the analysis of molecular diversity in the wild. A number of molecular ecology research groups therefore may switch from DNA-based to RNA-based typing in the near future. Sample preparation from natural populations, however, requires specific care and protocols when RNA is the target. Furthermore, NGS sequencing of transcriptome requires high amount of good-quality RNA. Here we present the results of RNA extraction experiments from various samples of 39 animal species caught in the wild. We compared tissue preparation and storage conditions, evaluated and improved standard RNA extraction protocols, and achieved RNA yield and quality suitable for NGS in all cases. We derive general guidelines for the production of ready-to-sequence RNA in nonmodel animals sampled in the field.