Nucleic Acids Research Advance Access originally published online on January 7, 2009
Nucleic Acids Research 2009 37(3):e21; doi:10.1093/nar/gkn1027
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Nucleic Acids Research, 2009, Vol. 37, No. 3 e21
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Telomere length measurement by a novel monochrome multiplex quantitative PCR method
Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA
*To whom correspondence should be addressed. Tel: +1 801 585 5520; Fax: +1 801 581 7796; Email: rcawthon{at}genetics.utah.edu
Received October 3, 2008. Revised December 9, 2008. Accepted December 10, 2008.
The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).
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