RNA isolation method for single embryo transcriptome analysis in zebrafish.
BACKGROUND
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 confounded. This can irreversibly reduce the robustness, resolution, or 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 zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible.


RESULTS
We developed a single embryo RNA extraction method based on sample homogenization in liquid nitrogen, RNA extraction with phenol and column purification. Evaluation of this method showed that: the quality of the RNA was very good with an average RIN value of 8.3-8.9; the yield was always >/= 200 ng RNA per embryo; the method was applicable to all stages of zebrafish embryogenesis; the success rate was almost 100%; and the extracted RNA performed excellent in microarray experiments in that the technical variation was much lower than the biological variation.


CONCLUSIONS
Presented is a high-quality, robust RNA isolation method. Obtaining sufficient RNA from single embryos eliminates the necessity of sample pooling and its associated drawbacks. Although our RNA isolation method has been setup for transcriptome analysis in zebrafish, it can also be used for other model systems and other applications like (q)PCR and transcriptome sequencing.
MicroArray Department & Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, the Netherlands. t.m.breit@uva.nl.
Mark
M
de Jong
Han
H
Rauwerda
Oskar
O
Bruning
Jurgo
J
Verkooijen
Herman P
HP
Spaink
Timo M
TM
Breit