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In situ RT-PCR Optimized for Electron Microscopy Allows Description of Subcellular Morphology of Target
Laura Cubas-Nuñez1, María Duran-Moreno2, Jessica Castillo-Villalba1
1Multiple Sclerosis and Neural Regeneration Research Group, Fundación para la Investigación La Fe, Hospital Universitario y Politécnico La FeValencia, Spain.
Abstract:
In situ RT-PCR detects and amplifies mRNA (cDNA) while obtaining spatial information of gene expression. When the intended use is an ultrastructural analysis of morphology, the procedure may be technically challenging and quality of tissue dramatically altered by proteolytic digestion and extreme astringency and temperature conditions. We describe a low-damaging protocol of in situ RT-PCR combined to conventional electron microscopy that preserves fine morphology, increases sensitivity, and decreases costs and complexity associated to RNA probes.
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