Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation.
BACKGROUND
RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene and Tempus blood collection tubes, and each comes with their own RNA purification method. In both systems the blood is immediately lysed when collected into the tube and RNA stabilized using proprietary reagents. Both systems enable minimal blood handling procedures thus minimizing the risk of inducing changes in gene expression through blood handling or processing. Because the RNA purification steps could influence the total RNA pool, we examined the impact of RNA isolation, using the PAXgene or Tempus method, on gene expression profiles.


RESULTS
Using microarrays as readout of RNA from stimulated whole blood we found a common set of expressed transcripts in RNA samples from either PAXgene or Tempus. However, we also found several to be uniquely expressed depending on the type of collection tube, suggesting that RNA purification methods impact results of differential gene expression profiling. Specifically, transcripts for several known PHA-inducible genes, including IFNgamma, IL13, IL2, IL3, and IL4 were found to be upregulated in stimulated vs. control samples when RNA was isolated using the ABI Tempus method, but not using the PAXgene method (p < 0.01, FDR corrected). Sequenom Quantiative Gene Expression (QGE) (SanDiego, CA) measures confirmed IL2, IL4 and IFNgamma up-regulation in Tempus purified RNA from PHA stimulated cells while only IL2 was up-regulated using PAXgene purified (p < 0.05).


CONCLUSIONS
Here, we demonstrate that peripheral blood RNA isolation methods can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological cohorts. A modified method based upon the Tempus system was found to provide high yield, good post-hybridization array quality, low variability in expression measures and was shown to produce differential expression results consistent with the predicted immunologic effects of PHA stimulation.
University of California, San Francisco, Immune Tolerance Network, 3 Bethesda Metro Suite 400, Bethesda, MD 20814, USA. aasare@immunetolerance.org
Adam L
AL
Asare
Svetlana A
SA
Kolchinsky
Zhong
Z
Gao
Richard
R
Wang
Khadir
K
Raddassi
Katarzyna
K
Bourcier
Vicki
V
Seyfert-Margolis
D001800:Blood Specimen Collection / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010835:Phytohemagglutinins / Q000494:pharmacology; D012313:RNA / Q000097:blood* / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000276:immunology
D010835:Phytohemagglutinins; D012313:RNA