Modified multiplex real-time PCR for quantification of differently sized cell-free dna fragments

E. A. Sokolova, I. V. Khlistun, D. N. Kushlinsky

    Research output: Contribution to journalArticlepeer-review

    Abstract

    There is evidence that size distribution of cell-free DNA (cfDNA) fragments can be diagnostically relevant. The present work describes a multiplex quantitative real-time polymerase chain reaction technique modified and validated by the authors to study the degree of cfDNA fragmentation in blood plasma. Based on the detection of Alu and hLINE-1 repeats, this technique employs fluorescent probes. We selected suitable primers and probes, optimized PCR conditions and estimated the dynamic range and sensitivity threshold of the assay. The modified PCR had a dynamic range of 6 logs, its efficiency being over 90 %. We demonstrated that cfDNA fragmentation index did not differ significantly between healthy women (n = 16) and women with stage III–IV ovarian cancer (n = 14). Therefore, further research on a larger sample is needed using electrophoretic cfDNA fractionation.

    Original languageEnglish
    Pages (from-to)19-23
    Number of pages5
    JournalBulletin of Russian State Medical University
    Volume6
    Issue number4
    DOIs
    Publication statusPublished - 1 Jul 2017

    Keywords

    • Alu repeats
    • Cell-free DNA
    • CfDNA
    • Fluorescent probe
    • HLINE-1 repeats
    • Multiplex quantitative PCR
    • Primers

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