Leukocyte Dynamics Influence Reference Gene Stability in Whole Blood: Data-Driven qRT-PCR Normalization Is a Robust Alternative for Measurement of Transcriptional Biomarkers

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Background: The use of reference genes for normalization of whole blood qRT-PCR data may be problematic in conditions such as stroke which induce alterations in white blood cell differential. In this study, we assessed the influence of stroke on the stability of commonly employed reference genes, and we evaluated data-driven normalization as an alternative. Methods: Peripheral whole blood was sampled from 33 stroke patients and 29 controls, and qRT-PCR was used to measure the expression levels of 10 target genes whose transcripts are known stroke biomarkers. Target gene expression levels were normalized via those of 2 frequently cited reference genes (ACTB and B2M) as well as with the NORMA-Gene data-driven normalization algorithm. Results: Whole blood expression levels of reference genes were significantly altered in stroke patients relative to controls. In comparison to normalization via reference genes, NORMA-Gene produced more robust target gene expression data in terms of differential expression dynamics, variance properties, and diagnostic performance. Conclusions: Our findings suggest that whole blood expression levels of commonly used reference genes may be sensitive to changes in white blood cell differential, and that data-driven qRT-PCR normalization approaches offer a powerful alternative.