Abstract
qPCR tomography was developed to study mRNA localization in complex biological samples that are embedded and cryo-sectioned. After total RNA extraction and reverse transcription, the spatial profiles of mRNAs and other functional RNAs were determined by qPCR.
The Xenopus laevis oocyte was selected as model, because of its large size (more than 1 mm) and large amount of total RNA ( 5 μg). Fifteen sections along the animal–vegetal axis were cut and prepared for quantification of 31 RNA targets using the high-throughput real-time RT-PCR (qPCR) BioMark™ platform. mRNAs were found to have two localization patterns, animal/central or vegetal.
Because of the high resolution in sectioning, it was possible to distinguish two subgroups of the vegetal gene patterns: germ plasm determinant pattern and profile of other vegetal genes.