INTRODUCTIONThis protocol describes how to isolate total RNA from amphioxus using a patented TRI reagent that is commercially available from several sources. The protocol can be applied to both adult tissues and embryos, although we recommend different sample pretreatment methods for adults and embryos. After the samples are prepared, they are homogenized in TRI reagent, which disrupts the cells and maintains the integrity of RNA. After the addition of chloroform and phase separation, RNA is extracted in the aqueous phase and subsequently recovered by precipitation with isopropyl alcohol. The resulting RNA can be used for gel blotting (Northern analysis), selection of polyadenylated RNA, or for synthesizing cDNA with reverse transcriptase PCR (RT-PCR).