title: Improving qPCR telomere length assays: Controlling for well position effects increases statistical power.
abstract: OBJECTIVE
  Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although, easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements.
  
  
  METHODS
  qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age, and between mother and offspring are examined.
  
  
  RESULTS
  First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error as compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity in our study.
  
  
  CONCLUSIONS
  Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, rerunning analyses of previous results with well position correction could serve as an independent test of the validity of these results.
authors.affiliation: Department of Anthropology, University of Washington, Seattle, Washington.|Center for Studies in Demography and Ecology, University of Washington, Seattle, Washington.
authors.forename: Dan T A
authors.identifier: http://orcid.org/0000-0003-0812-1862
authors.initials: DT
authors.lastname: Eisenberg
authors.affiliation: Department of Anthropology, Northwestern University, Evanston, Illinois.|Institute for Policy Research, Northwestern University, Evanston, Illinois.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Kuzawa
authors.affiliation: Department of Anthropology, Northwestern University, Evanston, Illinois.|Division of Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois.|Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
authors.forename: M Geoffrey
authors.initials: MG
authors.lastname: Hayes
mesh_terms: D000293:Adolescent; D000328:Adult; D000367:Age Factors; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008957:Models, Genetic; D015233:Models, Statistical; D010679:Philippines; D016133:Polymerase Chain Reaction / Q000379:methods*; D016615:Telomere / Q000502:physiology*; D059505:Telomere Homeostasis; D055815:Young Adult