Display options
Cite
de Jong M, Rauwerda H, Bruning O, et al. RNA isolation method for single embryo transcriptome analysis in zebrafish. BMC Res Notes. 2010;3:73doi: 10.1186/1756-0500-3-73.
de Jong, M., Rauwerda, H., Bruning, O., Verkooijen, J., Spaink, H. P., & Breit, T. M. (2010). RNA isolation method for single embryo transcriptome analysis in zebrafish. BMC research notes, 373. https://doi.org/10.1186/1756-0500-3-73
de Jong, Mark, et al. "RNA isolation method for single embryo transcriptome analysis in zebrafish." BMC research notes vol. 3 (2010): 73. doi: https://doi.org/10.1186/1756-0500-3-73
de Jong M, Rauwerda H, Bruning O, Verkooijen J, Spaink HP, Breit TM. RNA isolation method for single embryo transcriptome analysis in zebrafish. BMC Res Notes. 2010 Mar 16;3:73. doi: 10.1186/1756-0500-3-73. PMID: 20233395; PMCID: PMC2845602.
Copy Download .nbib Format: NLM
Share it on

BMC Res Notes. 2010 Mar 16;3:73. doi: 10.1186/1756-0500-3-73.

RNA isolation method for single embryo transcriptome analysis in zebrafish.

BMC research notes

Mark de Jong, Han Rauwerda, Oskar Bruning, Jurgo Verkooijen, Herman P Spaink, Timo M Breit

Affiliations

  1. MicroArray Department & Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, the Netherlands. [email protected].

PMID: 20233395 PMCID: PMC2845602 DOI: 10.1186/1756-0500-3-73

Abstract

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.

FINDINGS: 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.

References

  1. Development. 2008 May;135(10):1735-43 - PubMed
  2. Zebrafish. 2005;2(4):269-83 - PubMed
  3. Nat Rev Genet. 2006 Jan;7(1):55-65 - PubMed
  4. Int J Dev Biol. 2009;53(5-6):835-50 - PubMed
  5. PLoS Genet. 2005 Aug;1(2):260-76 - PubMed
  6. BMC Genomics. 2009;10:80 - PubMed
  7. Physiol Genomics. 2005 Aug 11;22(3):346-55 - PubMed
  8. Dev Dyn. 2009 Jan;238(1):150-61 - PubMed
  9. Hum Mol Genet. 2009 Apr 15;18(R1):R107-12 - PubMed
  10. Mol Immunol. 2009 Jul;46(11-12):2317-32 - PubMed
  11. PLoS One. 2008;3(12):e4055 - PubMed
  12. Nucleic Acids Res. 2002 Jan 1;30(1):207-10 - PubMed
  13. Comp Biochem Physiol C Toxicol Pharmacol. 2004 Jul;138(3):363-73 - PubMed
  14. J Immunol. 2009 May 1;182(9):5641-53 - PubMed
  15. Nucleic Acids Res. 2005 Jan 1;33(Database issue):D562-6 - PubMed
  16. Comp Biochem Physiol C Toxicol Pharmacol. 2004 Jul;138(3):351-62 - PubMed
  17. Reproduction. 2008 May;135(5):581-92 - PubMed
  18. Nat Genet. 2003 Mar;33 Suppl:285-93 - PubMed
  19. Bioinformatics. 2007 Jul 1;23(13):i313-8 - PubMed
  20. Dev Dyn. 2009 Jul;238(7):1813-26 - PubMed

Publication Types