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    beta-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

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    Author
    Kraan, CM; Cornish, KM; Bui, QM; Li, X; Slater, HR; Godler, DE
    Date
    2018-02-23
    Source Title
    PLoS One
    Publisher
    PUBLIC LIBRARY SCIENCE
    University of Melbourne Author/s
    Kraan, Claudine; Bui, Quang; Slater, Howard; Godler, David
    Affiliation
    Melbourne School of Population and Global Health
    Paediatrics (RCH)
    Metadata
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    Document Type
    Journal Article
    Citations
    Kraan, C. M., Cornish, K. M., Bui, Q. M., Li, X., Slater, H. R. & Godler, D. E. (2018). beta-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies. PLOS ONE, 13 (2), https://doi.org/10.1371/journal.pone.0192151.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/255935
    DOI
    10.1371/journal.pone.0192151
    Abstract
    Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

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