Technical variation between RNA isolations from individual embryos

Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is lost for inclusion into statistical models used to analyze transcriptome data. This can irreversibly reduce the robustness, resolution and expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using the zebrafish as a model. Zebrafish embryos were selected from the germ ring stage. RNA from 4 individual embryos and RNA from 4 pooled embryos and afterwards splitted material was isolated. These 8 samples were hybridized against a common reference made from a pool of 20 embryos.