The role of non-coding RNAin developmental toxicity by dioxin

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1) Development of novel DOP-PCR method with transcriptome as a target: We developed a novel degenerate oligonucleotide-primed PCR (DOP-PCR) with high reproducibility. We used mono-primers for DOP-PCR, as there were many sets of inverse-repeat sequence adequate for PCR reaction in transcriptome of zebrafish (Danio rerio). Primed part of primers (specific sequence part) was designed to obtain about 10 bands for each PCR reaction, using our developed computer program.2) Differential display approach in search for transcripst increased by TCDD : We carried out differential display based on DOP-PCR described above and found CYP1C1 and 1C2 were dramatically upregulated by TCDD exposure. Gene knockdown of CYP1C2 rescued mesencephalic circulation from TCDD-induced failure, suggesting the possible rule of CYP1C2 in TCDD developmental toxicity.3) Application of DOP-PCR for other fish species with zebrafish primers: DOP-PCR with the primers designed for zebrafish could be used for reproducible PCR products with medaka (Oryzias latips) and other 8 fish species. The same strategy could be applicable for various mammals or birds using DOP-PCR primers designed for rodents and domestic fowls.4) Effects of dioxin on miRNA transcriptional status: Microarray media with 272 different miRNA spots was used to clarify the effect of TCDD on miRNA transcriptinal status. About 10 miRNA species showed two-fold changes or more in trasciptational level by TCDD exposure at 48 and 72 hours post fertilization, respectively. Most of these miRNA speceis overlap between two stages.