Whole blood and leukocyte RNA isolation for gene expression analyses

Abstract

The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases

“…In the last two years, the demand for microarray-based gene expression analysis 17,18 has driven the development of new methods for harvesting and preserving RNA. To date, these methods have not been exploited for genetic testing, although their potential is clear.…”

Genome-wide discovery and verification of novel structured RNAs inPlasmodium falciparum

“…RBCs have been demonstrated to carry large amounts of globin RNA, which affects human microarray analysis (Feezor et al 2004). Additionally, we and others have found that uninfected RBCs contain abundant human miRNAs (Rathjen et al 2006;our observations [unpubl.]).…”

Development of a Genomic Metric that can be Rapidly Used to Predict Clinical Outcome in Severely Injured Trauma Patients

“…Samples were collected with signed informed consent; each institution had their own IRB approval, and de-identified clinical data are now in the public domain (16). Blood samples were collected within 12 hours of injury and at 1, 4, 7, 14, 21, and 28 days after the injury while hospitalized in the intensive care unit, whole blood nucleated cells were isolated, and genome-wide expression analyses of 54,675 probe sets were performed using the Affymetrix U133 GeneChip ™ (17,18).…”