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Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types

The Pharmacogenomics Journal volume 4, pages 193–207 (2004)Cite this article

ABSTRACT

Owing to its clinical accessibility, peripheral blood is probably the best source for the assessment of differences or changes in gene expression associated with disease or drug response and therapy. Gene expression patterns in peripheral blood cells greatly depend on temporal and interindividual variations. However, technical aspects of blood sampling, isolation of cellular components, RNA isolation techniques and clinical aspects such as time to analysis and temperature during processing have been suggested to affect gene expression patterns. We therefore assessed gene expression patterns in peripheral blood from 29 healthy individuals by using Affymetrix microarrays. When RNA isolation was delayed for 20–24 h—a typical situation in clinical studies—gene signatures related to hypoxia were observed, and downregulation of genes associated with metabolism, cell cycle or apoptosis became dominant preventing the assessment of gene signatures of interindividual variation. Similarly, gene expression patterns were strongly dependent on choice of cell and RNA isolation and preparation techniques. We conclude that for large clinical studies, it is crucial to reduce maximally the time to RNA isolation. Furthermore, prior to study initiation, the cell type of interest should already be defined. Our data therefore will help to optimize clinical studies applying gene expression analysis of peripheral blood to exploit drug responses and to better understand changes associated with disease.

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Abbreviations

BD:

BD-CPT

BUFFY:

buffy-coat samples

FI-8C:

samples prepared by Ficoll at 8°C

FI-DELAYED:

samples with delayed preparation by Ficoll

IL:

interleukin

PBMC:

peripheral blood mononuclear cells

PM:

perfect match model

PAX:

PAX gene

RT:

room temperature

SLE:

systemic lupus erythematosus

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Acknowledgements

We are grateful to all blood donors for donating blood for this study. We thank David Finkelstein and John Martin from the Data Analysis Team, Affymetrix Genomics Collaborations, Marc Beyer, and Jürgen Wolf for helpful discussions and data analysis throughout the project. This work was supported in part by a Sofja Kovalevskaja award from the Alexander von Humboldt-Foundation (JLS), a fellowship by the Frauke-Weiskam Foundation (TZ) and a stipend for graduate students from the Köln Fortune program of the University Hospital Cologne (US).

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  1. S Debey and U Schoenbeck: Both these authors contributed equally to this work

Authors and Affiliations

  1. Molecular Tumor Biology and Tumor Immunology, Center for Internal Medicine, University of Cologne, Cologne, Germany

    S Debey, U Schoenbeck, T Zander & J L Schultze

  2. Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Cologne, Germany

    M Hellmich

  3. Division of Transfusion Medicine, University of Cologne, Cologne, Germany

    B S Gathof

  4. Genomics Collaborations, Affymetrix Inc., Santa Clara, CA, USA

    R Pillai

  5. Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany

    J L Schultze

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Correspondence to J L Schultze.

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Debey, S., Schoenbeck, U., Hellmich, M. et al. Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types. Pharmacogenomics J 4, 193–207 (2004). https://doi.org/10.1038/sj.tpj.6500240

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