Whole blood and leukocyte RNA isolation for gene expression analyses

被引:157
作者
Feezor, RJ
Baker, HV
Mindrinos, M
Hayden, D
Tannahill, CL
Brownstein, BH
Fay, A
MacMillan, S
Laramie, J
Xiao, WZ
Moldawer, LL
Cobb, JP
Laudanski, K
Miller-Graziano, CL
Maier, RV
Schoenfeld, D
Davis, RW
Tompkins, RG
机构
[1] Harvard Univ, Massachusetts Gen Hosp, Sch Med,Dept Surg, Trauma Serv, Boston, MA 02114 USA
[2] Univ Florida, Coll Med, Dept Surg, Gainesville, FL USA
[3] Univ Florida, Coll Med, Dept Microbiol & Mol Genet, Gainesville, FL USA
[4] Stanford Genome Technol Ctr, Palo Alto, CA USA
[5] Shriners Burn Ctr, Dept Surg, Boston, MA USA
[6] Washington Univ, Sch Med, Dept Surg, St Louis, MO 63110 USA
[7] MIT, Dept Chem Engn, Cambridge, MA 02139 USA
[8] Univ Rochester, Sch Med & Dent, Dept Surg, Rochester, NY USA
[9] Univ Washington, Harborview Med Ctr, Dept Surg, Seattle, WA 98104 USA
关键词
microarray; buffy coat; PAXgene; inflammation; trauma;
D O I
10.1152/physiolgenomics.00020.2004
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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.
引用
收藏
页码:247 / 254
页数:8
相关论文
共 17 条
[1]  
Anderson Robert N, 2003, Natl Vital Stat Rep, V52, P1
[2]   Comparison of microarray designs for class comparison and class discovery [J].
Dobbin, K ;
Simon, R .
BIOINFORMATICS, 2002, 18 (11) :1438-1445
[3]   Cluster analysis and display of genome-wide expression patterns [J].
Eisen, MB ;
Spellman, PT ;
Brown, PO ;
Botstein, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (25) :14863-14868
[4]  
Fleiss J., 1986, Reliability of measurement: the design and analysis of clinical experiments
[5]   Transcript profiling of human platelets using microaray and serial analysis of gene expression [J].
Gnatenko, DV ;
Dunn, JJ ;
McCorkle, SR ;
Weissmann, D ;
Perrotta, PL ;
Bahou, WF .
BLOOD, 2003, 101 (06) :2285-2293
[6]   Molecular classification of cancer: Class discovery and class prediction by gene expression monitoring [J].
Golub, TR ;
Slonim, DK ;
Tamayo, P ;
Huard, C ;
Gaasenbeek, M ;
Mesirov, JP ;
Coller, H ;
Loh, ML ;
Downing, JR ;
Caligiuri, MA ;
Bloomfield, CD ;
Lander, ES .
SCIENCE, 1999, 286 (5439) :531-537
[7]   Guidelines - Expression profiling - best practices for data generation and interpretation in clinical trials [J].
Hoffman, EP ;
Awad, T ;
Palma, J ;
Webster, T ;
Hubbell, E ;
Warrington, JA ;
Spirais, A ;
Wright, G ;
Buckley, J ;
Triche, T ;
Davis, R ;
Tibshirani, R ;
Xiao, WH ;
Jones, W ;
Tompkins, R ;
West, M .
NATURE REVIEWS GENETICS, 2004, 5 (03) :229-237
[8]   Oligonucleotide microarray for prediction of early intrahepatic recurrence of hepatocellular carcinoma after curative resection [J].
Iizuka, N ;
Oka, M ;
Yamada-Okabe, H ;
Nishida, M ;
Maeda, Y ;
Mori, N ;
Takao, T ;
Tamesa, T ;
Tangoku, A ;
Tabuchi, H ;
Hamada, K ;
Nakayama, H ;
Ishitsuka, H ;
Miyamoto, T ;
Hirabayashi, A ;
Uchimura, S ;
Hamamoto, Y .
LANCET, 2003, 361 (9361) :923-929
[9]   Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection [J].
Li, C ;
Wong, WH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (01) :31-36
[10]   Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization [J].
Müller, MC ;
Merx, K ;
Weisser, A ;
Kreil, S ;
Lahaye, T ;
Hehlmann, R ;
Hochhaus, A .
LEUKEMIA, 2002, 16 (12) :2395-2399