cDNA targets improve whole blood gene expression profiling and enhance detection of pharmocodynamic biomarkers: a quantitative platform analysis.
BACKGROUND
Genome-wide gene expression profiling of whole blood is an attractive method for discovery of biomarkers due to its non-invasiveness, simple clinical site processing and rich biological content. Except for a few successes, this technology has not yet matured enough to reach its full potential of identifying biomarkers useful for clinical prognostic and diagnostic applications or in monitoring patient response to therapeutic intervention. A variety of technical problems have hampered efforts to utilize this technology for identification of biomarkers. One significant hurdle has been the high and variable concentrations of globin transcripts in whole blood total RNA potentially resulting in non-specific probe binding and high background. In this study, we investigated and quantified the power of three whole blood profiling approaches to detect meaningful biological expression patterns.


METHODS
To compare and quantify the impact of different mitigation technologies, we used a globin transcript spike-in strategy to synthetically generate a globin-induced signature and then mitigate it with the three different technologies. Biological differences, in globin transcript spiked samples, were modeled by supplementing with either 1% of liver or 1% brain total RNA. In order to demonstrate the biological utility of a robust globin artifact mitigation strategy in biomarker discovery, we treated whole blood ex vivo with suberoylanilide hydroxamic acid (SAHA) and compared the overlap between the obtained signatures and signatures of a known biomarker derived from SAHA-treated cell lines and PBMCs of SAHA-treated patients.


RESULTS
We found cDNA hybridization targets detect at least 20 times more specific differentially expressed signatures (2597) between 1% liver and 1% brain in globin-supplemented samples than the PNA (117) or no treatment (97) method at FDR = 10% and p-value < 3x10-3. In addition, we found that the ex vivo derived gene expression profile was highly concordant with that of the previously identified SAHA pharmacodynamic biomarkers.


CONCLUSIONS
We conclude that an amplification method for gene expression profiling employing cDNA targets effectively mitigates the negative impact on data of abundant globin transcripts and greatly improves the ability to identify relevant gene expression based pharmacodynamic biomarkers from whole blood.
Parrish
Mark L
ML
Covance Genomics Laboratory, LLC, Seattle, WA 98109, USA. mark.parrish@covance.com
Wright
Chris
C
Rivers
Yarek
Y
Argilla
David
D
Collins
Heather
H
Leeson
Brendan
B
Loboda
Andrey
A
Nebozhyn
Michael
M
Marton
Matthew J
MJ
Lejnine
Serguei
S
D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000097:blood*
D018076:DNA, Complementary; D012313:RNA