Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: de Jong, Mark, Rauwerda, Han, Bruning, Oskar, Verkooijen, Jurgo, Spaink, Herman P, Breit, Timo M
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845602/
https://www.ncbi.nlm.nih.gov/pubmed/20233395
http://dx.doi.org/10.1186/1756-0500-3-73
_version_ 1782179416669945856
author de Jong, Mark
Rauwerda, Han
Bruning, Oskar
Verkooijen, Jurgo
Spaink, Herman P
Breit, Timo M
author_facet de Jong, Mark
Rauwerda, Han
Bruning, Oskar
Verkooijen, Jurgo
Spaink, Herman P
Breit, Timo M
author_sort de Jong, Mark
collection PubMed
description 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.
format Text
id pubmed-2845602
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-28456022010-03-26 RNA isolation method for single embryo transcriptome analysis in zebrafish de Jong, Mark Rauwerda, Han Bruning, Oskar Verkooijen, Jurgo Spaink, Herman P Breit, Timo M BMC Res Notes Technical Note 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. BioMed Central 2010-03-16 /pmc/articles/PMC2845602/ /pubmed/20233395 http://dx.doi.org/10.1186/1756-0500-3-73 Text en Copyright ©2010 de Jong et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Technical Note
de Jong, Mark
Rauwerda, Han
Bruning, Oskar
Verkooijen, Jurgo
Spaink, Herman P
Breit, Timo M
RNA isolation method for single embryo transcriptome analysis in zebrafish
title RNA isolation method for single embryo transcriptome analysis in zebrafish
title_full RNA isolation method for single embryo transcriptome analysis in zebrafish
title_fullStr RNA isolation method for single embryo transcriptome analysis in zebrafish
title_full_unstemmed RNA isolation method for single embryo transcriptome analysis in zebrafish
title_short RNA isolation method for single embryo transcriptome analysis in zebrafish
title_sort rna isolation method for single embryo transcriptome analysis in zebrafish
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845602/
https://www.ncbi.nlm.nih.gov/pubmed/20233395
http://dx.doi.org/10.1186/1756-0500-3-73
work_keys_str_mv AT dejongmark rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish
AT rauwerdahan rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish
AT bruningoskar rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish
AT verkooijenjurgo rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish
AT spainkhermanp rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish
AT breittimom rnaisolationmethodforsingleembryotranscriptomeanalysisinzebrafish