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Effects of Storage, RNA Extraction, Genechip Type, and Donor Sex on Gene Expression Profiling of Human Whole Blood

¹ National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC.
² Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC.
³ Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC.

^aAddress correspondence to this author at: National Center for Computational Toxicology (D343-03), Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27711. Fax 919-541-1194; e-mail dix.david{at}epa.gov.

Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability.

Methods: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables.

Results: Storage of blood samples for >1 week at 4 °C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex.

Conclusion: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

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