title: Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods.
abstract: BACKGROUND
  Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay.
  
  
  RESULTS
  Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays.
  
  
  CONCLUSIONS
  Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.
authors.affiliation: Evolutionary Genetics Group, Department of Genetics, Microbiology, and Toxicology, Stockholm University, Stockholm, S-106 91, Sweden. mortentolsen@gmail.com
authors.forename: Morten Tange
authors.initials: MT
authors.lastname: Olsen
authors.forename: Martine
authors.initials: M
authors.lastname: Bérubé
authors.forename: Jooke
authors.initials: J
authors.lastname: Robbins
authors.forename: Per J
authors.initials: PJ
authors.lastname: Palsbøll
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000378:metabolism; D050640:Humpback Whale / Q000235:genetics* / Q000378:metabolism; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D016615:Telomere / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers