Mitochondrial DNA Analysis for Cell Authentication

Mitochondrial DNA Analysis for Cell Authentication

(RUO = For Research Use Only)

  • Human cultured cell lines are used in a number of biomedical research and clinical applications, including cancer research, drug discovery, genetics and biobanking. However, misidentified human and animal cell lines have continued to be used even today despite multiple and repeated warnings, articles and letters by prominent scientists in the field calling for authentication (1-4). In some cases, these cross contaminants have been carried along for decades (2). Furthermore, it has been demonstrated that cells maintained in culture and subcultured again and again tend to evolve – a process that is often referred to as “genetic drift” (5).
  • A variety of methods and markers are currently used to check the identity and purity of cultured cells (3, 6), but up to now, none have utilized sequence analysis of mitochondrial DNA (7, 8) as a tool to detect cross-contamination. Mitochondrial DNA has a high mutation rate and is present in hundreds or even thousands of copies in mammalian cells (9). Cell lines, therefore, have very specific mtDNA sequences that serve as “mtDNA markers” which in turn can be used to trace potential contaminants. Every cell line that is newly established or that is brought into the laboratory should have its mtDNA sequenced to establish a reference sequence. mtDNA of cells passaged, subcultured or subcloned can then be sequenced again in the future to verify the cell line’s identity, lack of mtDNA changes and lack of cross-contaminants. Mitochondrial genome sequencing identifies highly discriminative mtDNA markers that can then be used not only for the detection and identification of contaminating human cells, but also for cell line authentication.
  • Genes (37): MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, MT-TY, MT-RNR1, and MT-RNR2
    Test Code: 8101 or 8102
    Clinical Indications:
    • Cell line contamination check
    • Authentication and clonality of cell lines
    Test Info Sheet: Mitochondrial DNA Analysis for Cell Authentication
    Requisition: Mitochondrial Test Requisition Form and/or Contact ApolloGen Diagnostic Laboratory
  • Turn-Around Time: 2 Weeks
    Specimen Requirement:
    • Cell Pellet: 100,000 cells provided as a frozen cell pellet shipped on dry ice
    • DNA: 500 ng of total DNA (at 25 ng/µL or higher concentration) in water or TE shipped on frozen ice packs
    • Blood: 3-5 mL blood (1 mL minimum in a Lavender Top / EDTA Tube) shipped at room temperature
    Other Specimens: Contact ApolloGen Diagnostic Laboratory
  • Test Results: Results typically are provided as a table that lists the variants and heteroplasmies found
    Pricing: Please contact us at (949) 916-8886 or inquiries@apollogen.com for current pricing
  • Methodology: Long-range PCR followed by Next-Generation Sequencing (NGS)
    Related Tests:
    iPSC Mitochondrial DNA Analysis (RUO)
    Comprehensive Mitochondrial Genome Analysis
  • References:
    1. Kniss, D. A. & Summerfield, T. L. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research. Reprod. Sci. Thousand Oaks Calif 21, 1015–1019 (2014).
    2. Jäger, W. et al. Hiding in plain view: genetic profiling reveals decades old cross contamination of bladder cancer cell line KU7 with HeLa. J. Urol. 190, 1404–1409 (2013).
    3. Johnen, G. et al. Cross-contamination of a UROtsa stock with T24 cells–molecular comparison of different cell lines and stocks. PloS One 8, e64139 (2013)
    4. Marx, V. Cell-line authentication demystified. Nat Methods 11, 483-488 (2014).
    5. Torsvik, A. et al. U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells. Cancer Med. 3, 812–824 (2014).
    6. Baghbaderani, B.A. et al. Detailed characterization of human induced pluripotent stem cells manufactured for therapeutic applications. Stem Cell Rev 12, 394-420 (2016).
    7. Tang, S. & Huang, T. Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system. Biotechniques 48, 287–296 (2010).
    8. Huang, T. Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy. Curr Protoc Hum Genet Chapter 19, Unit19.8 (2011).
    9. Wallace, D. C. & Chalkia, D. Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring Harb. Perspect. Biol. 5, a021220 (2013).
    10. http://www.mitomap.org/MITOMAP

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