title: 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.
authors.affiliation: Department of Surgery, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Feezor
authors.forename: Henry V
authors.initials: HV
authors.lastname: Baker
authors.forename: Michael
authors.initials: M
authors.lastname: Mindrinos
authors.forename: Doug
authors.initials: D
authors.lastname: Hayden
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Tannahill
authors.forename: Bernard H
authors.initials: BH
authors.lastname: Brownstein
authors.forename: Adrian
authors.initials: A
authors.lastname: Fay
authors.forename: Sandra
authors.initials: S
authors.lastname: MacMillan
authors.forename: Jason
authors.initials: J
authors.lastname: Laramie
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Xiao
authors.forename: Lyle L
authors.initials: LL
authors.lastname: Moldawer
authors.forename: J Perren
authors.initials: JP
authors.lastname: Cobb
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Laudanski
authors.forename: Carol L
authors.initials: CL
authors.lastname: Miller-Graziano
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Maier
authors.forename: David
authors.initials: D
authors.lastname: Schoenfeld
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Tompkins
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000378:metabolism; D001800:Blood Specimen Collection / Q000592:standards; D016000:Cluster Analysis; D004768:Enterotoxins / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards* / Q000706:statistics & numerical data; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000592:standards* / Q000706:statistics & numerical data; D012313:RNA / Q000097:blood* / Q000235:genetics*; D015203:Reproducibility of Results; D012107:Research Design / Q000592:standards; D013048:Specimen Handling / Q000592:standards; D014947:Wounds and Injuries / Q000097:blood
chemical_list: D000942:Antigens, Bacterial; D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal; D012313:RNA