Selective Depletion of Ribosomal RNA from Total RNA for Next-generation Sequencing Analysis of Maternal mRNA in Eggs of the Japanese Eel Anguilla japonica
泉, ひかり ,
萩原, 聖士 玄, 浩一郎 ,
井尻, 成保 ,
130 , 2015-12-20 , 北海道大学大学院水産科学研究院
Removal of ribosomal RNAs(rRNA) from total RNA in eggs before preparation of a RNA library is required for the analysis of maternal mRNA using next-generarion sequencing ; this mRNA is essential for normal development. In mammals, the rRNA-depletion method is well-established and is already being used for the development of experiment kits. However, this method is not designed for other species, e.g., non-model organisms. In the current study, we attempted to remove rRNA from total RNA of Japanese eel Anguilla japonica, which is one of the most important aquaculture species in Japan. Removal was performed via hibridization-selection using mammalian probes from a commmercially available kit and/or an ordinally designed eel probe. Quantitative polymerase chain reaction(PCR) was performed to examine the depletion efficiency of 18S rRNA and 28S rRNA were comparative between each method, and >98% depletion was achieved. In addition, RNA-sequencing analysis using a next-generation sequencer and read-mapping analysis were carried out using the rRNA-depleted RNA, which was prepared using a mammalian probe, and mapping analysis revealed that rRNA reads were <1% of the total reads. These results suggest that an rRNA-depletion kit designed for a mammal can also be used for fish species.