Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte.
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.
Sindelka
Radek
R
Laboratory of Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic.
Jonák
Jiri
J
Hands
Rebecca
R
Bustin
Stephen A
SA
Kubista
Mikael
M
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013997:Time Factors; D014054:Tomography / Q000379:methods*; D014982:Xenopus laevis
D012333:RNA, Messenger