A minimum variance method for genome-wide data-driven normalization of quantitative real-time polymerase chain reaction expression data.
Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of nondetection (∼50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods.
Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO 80206, USA; Computational Bioscience Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA.
Benjamin
B
Garcia
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA.
Nicholas D
ND
Walter
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94304, USA.
Gregory
G
Dolganov
Department of Health Research and Policy, Stanford University, Stanford, CA 94305, USA.
Marc
M
Coram
Division of Pulmonary & Critical Care Medicine, San Francisco General Hospital, University of California, San Francisco, San Francisco, CA, USA.
J Lucian
JL
Davis
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94304, USA.
Gary K
GK
Schoolnik
Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO 80206, USA; Computational Bioscience Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address: StrongM@NJHealth.org.
Michael
M
Strong
D016680:Genome, Bacterial; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D012313:RNA / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology
D012313:RNA
Data-driven normalization; Multiplex qRT-PCR