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| Status |
Public on Jan 30, 2009 |
| Title |
Gene Expression Profiling of Whole Blood: Comparison of target preparation methods |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system.
Results: We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples.
Conclusion: RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles.
Keywords: Whole Blood, Protocol Variation, Expression Profiling, Microarray, globin, PAXGene, PNA
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| Overall design |
Blood samples were collected from healthy, human donors in PAXGene tubes; RNA was isolated either on day of collection or after freezing and storage. Four different methods of microarray target preparation for whole blood RNA samples were examined. Three of the methods used total RNA extracted from whole blood as the starting sample for mRNA amplification and target labeling: Affymetrix one-cycle target labeling (Method 1: no depletion_Affymetrix); Affymetrix one-cycle target labeling with globin PNAs added during cDNA synthesis (Method 2: Globin PNAs_Affymetrix); and NuGEN Ovation system v1 (Method 4: no depletion_NuGEN). In the other method tested, total RNA was treated with Ambion GLOBINclear to reduce globin transcripts prior to labeling with the Affymetrix one-cycle labeling protocol (Method 3: GLOBINclear_Affymetrix). The Affymetrix one-cycle target labeling protocol produces biotin-labeled, amplified cRNA; the NuGEN protocol produces biotin-labeled, amplified cDNA targets. The analysis of this study included 24 total samples, with three biological replicates per sample storage state (fresh/frozen) and labeling method (Methods 1 - 4).
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| Contributor(s) |
Vartanian K, Slottke R, Johnstone T, Casale A, Planck SR, Choi D, Smith JR, Rosenbaum JT, Harrington CA |
| Citation(s) |
19123946 |
| Submission date |
Oct 21, 2008 |
| Last update date |
Jul 08, 2016 |
| Contact name |
Christina A Harrington |
| E-mail(s) |
harringc@ohsu.edu
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| Phone |
503-418-2737
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| Fax |
503-418-9381
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| Organization name |
Oregon Health and Science University
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| Department |
Gene Microarray Shared Resource
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| Lab |
Affymetrix Microarray Core Facility
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| Street address |
3303 SW Bond Ave
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| City |
Portland |
| State/province |
OR |
| ZIP/Postal code |
97239 |
| Country |
USA |
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| Platforms (1) |
| GPL201 |
[HG-Focus] Affymetrix Human HG-Focus Target Array |
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| Samples (24)
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| Relations |
| BioProject |
PRJNA109619 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE13292_RAW.tar |
20.2 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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