title: Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real-Time Quantitative PCR on the LightCycler 480: Sources of Variability and Quality Control Considerations.
abstract: Telomere length (TL) measurement is central to many biomedical research, population, and epidemiology studies, with promising potential as a clinical tool. Various assays are used to determine TL, depending on the type and size of the sample. We describe the detailed optimization of a monochromatic multiplex real-time quantitative PCR (MMqPCR) assay for relative TL using the LightCycler 480. MMqPCR was initially developed using a different instrument with many separate reagents. Differences in instrument performance, reagents, and workflow required substantial optimization for the assay to be compatible with the LightCycler 480. We optimized the chemistry of the assay using a purchased one-component reaction mix and herein describe sources of variability and quality control relevant to the MMqPCR TL assay on any instrument. Finally, the assay was validated against other TL assays, such as terminal restriction fragment, Southern blot, and flow fluorescent in situ hybridization. The correlations obtained between data from MMqPCR and these assays (R(2) = 0.88 and 0.81) were comparable to those seen with the monoplex version (R(2) = 0.85 and 0.82) when the same samples were assayed. The intrarun and interrun CV ranged from 4.2% to 6.2% and 3.2% to 4.9%, respectively. We describe a protocol for measuring TL on the LightCycler platform that provides a robust high-throughput method applicable to clinical diagnostics or large-scale studies of archived specimens.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Anthony Y Y
authors.initials: AYY
authors.lastname: Hsieh
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Sara
authors.initials: S
authors.lastname: Saberi
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Ajaykumar
authors.affiliation: Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Kyle
authors.initials: K
authors.lastname: Hukezalie
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Izabella
authors.initials: I
authors.lastname: Gadawski
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Beheroze
authors.initials: B
authors.lastname: Sattha
authors.affiliation: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: helene.cote@ubc.ca.
authors.forename: Hélène C F
authors.initials: HCF
authors.lastname: Côté
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016615:Telomere / Q000235:genetics*; D059505:Telomere Homeostasis / Q000235:genetics*