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Nucleic Acids Research Advance Access published online on November 26, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm1024
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte

Radek Sindelka1, Jiri Jonák1,4, Rebecca Hands2, Stephen A. Bustin2 and Mikael Kubista1,3,*

1Laboratory of Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic, 2Barts and the London Queen Mary's School of Medicine and Dentistry, Academic Surgery, London, E1 1BB, UK, 3TATAA Biocenter, Medicinargatan 8A, 413 46 Göteborg, Sweden and 4Institute of Medical Biochemistry, First Medical Faculty, Charles University, Katerinska 1083, 12000 Prague 2, Czech Republic

*To whom correspondence should be addressed. Tel: +46 31 741 18 00; Fax: +46 31 741 17 01; Email: mikael.kubista{at}img.cas.cz

Received October 9, 2007. Revised October 27, 2007. Accepted October 28, 2007.

Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal–vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.


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