Whole blood and leukocyte RNA isolation for gene expression analyses.
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.
Feezor
Robert J
RJ
Department of Surgery, University of Florida College of Medicine, Gainesville, Florida, USA.
Baker
Henry V
HV
Mindrinos
Michael
M
Hayden
Doug
D
Tannahill
Cynthia L
CL
Brownstein
Bernard H
BH
Fay
Adrian
A
MacMillan
Sandra
S
Laramie
Jason
J
Xiao
Wenzhong
W
Moldawer
Lyle L
LL
Cobb
J Perren
JP
Laudanski
Krzysztof
K
Miller-Graziano
Carol L
CL
Maier
Ronald V
RV
Schoenfeld
David
D
Davis
Ronald W
RW
Tompkins
Ronald G
RG
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
D000942:Antigens, Bacterial; D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal; D012313:RNA