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Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos
BACKGROUND: The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast,...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2007
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2246118/ https://www.ncbi.nlm.nih.gov/pubmed/18067662 http://dx.doi.org/10.1186/1472-6750-7-87 |
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author | Hartshorn, Cristina Eckert, Judith J Hartung, Odelya Wangh, Lawrence J |
author_facet | Hartshorn, Cristina Eckert, Judith J Hartung, Odelya Wangh, Lawrence J |
author_sort | Hartshorn, Cristina |
collection | PubMed |
description | BACKGROUND: The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. RESULTS: We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. CONCLUSION: This paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics. |
format | Text |
id | pubmed-2246118 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-22461182008-02-19 Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos Hartshorn, Cristina Eckert, Judith J Hartung, Odelya Wangh, Lawrence J BMC Biotechnol Methodology Article BACKGROUND: The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. RESULTS: We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. CONCLUSION: This paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics. BioMed Central 2007-12-07 /pmc/articles/PMC2246118/ /pubmed/18067662 http://dx.doi.org/10.1186/1472-6750-7-87 Text en Copyright © 2007 Hartshorn 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 | Methodology Article Hartshorn, Cristina Eckert, Judith J Hartung, Odelya Wangh, Lawrence J Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title | Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title_full | Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title_fullStr | Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title_full_unstemmed | Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title_short | Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos |
title_sort | single-cell duplex rt-late-pcr reveals oct4 and xist rna gradients in 8-cell embryos |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2246118/ https://www.ncbi.nlm.nih.gov/pubmed/18067662 http://dx.doi.org/10.1186/1472-6750-7-87 |
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