Efficiency clustering for low-density microarrays and its application to QPCR.

BMC Bioinformatics
Eric F LockDirk P Dittmer

Abstract

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency. Our results indicate that QPCR primer-pairs differ significantly both in reliability and efficiency. They can only be used in an array format if the raw data (so called CT values for real-time QPCR) are transformed to take these differences into account. We developed a novel method to obtain efficiency-adjusted CT values. We introduce transformed confidence intervals as a novel measure to identify unreliable primers. We introduce a robust clustering algorithm to combine efficiencies of groups of probes, and our results indicate that using n < 10 cluster-based mean efficiencies is comparable to using individually determined efficiency adjustments for each primer pair (N = 96-1024). Careful estimation of primer efficiency i...Continue Reading

References

Dec 9, 1998·Proceedings of the National Academy of Sciences of the United States of America·M B EisenD Botstein
Jul 11, 2003·Nucleic Acids Research·Stuart N PeirsonRussell G Foster
Aug 22, 2003·Nucleic Acids Research·Tzachi BarMikael Kubista
Feb 27, 2004·Nucleic Acids Research·Thomas D SchmittgenLiuqing Yang
Nov 30, 2005·Nucleic Acids Research·Chelsey HilscherDirk P Dittmer
Mar 1, 2006·BMC Bioinformatics·Joshua S YuanC Neal Stewart
Sep 15, 2006·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Jan H SchefeHeiko Funke-Kaiser
Apr 21, 2007·BMC Bioinformatics·Yann KarlenNicolas Mermod

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Citations

May 9, 2012·Wiley Interdisciplinary Reviews. RNA·Pauline Chugh, Dirk P Dittmer
Jan 27, 2015·Computers in Biology and Medicine·Oliver FaustU Rajendra Acharya

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Methods Mentioned

BETA
PCR

Software Mentioned

Eisen
R
R script Primer Efficiency Analysis
R environment
R programming environment for statistical computing and graphics
REST
R script
PEA

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