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  • Title: Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.
    Author: Theophile K, Jonigk D, Kreipe H, Bock O.
    Journal: Diagn Mol Pathol; 2008 Jun; 17(2):101-6. PubMed ID: 18382351.
    Abstract:
    The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type-specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA. Therefore, a successful amplification of cell-type-specific mRNA targets from FFPE tissues becomes more and more essential. To optimize the hitherto limited technical options, we applied 3 commercial amplification kits on FFPE single cells. We thereby determined the approach of target-specific cDNA amplification as being notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated.
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