Optimization of a high-throughput whole blood expression profiling methodology and its application to assess the pharmacodynamics of interferon (IFN) beta-1a or polyethylene glycol-conjugated IFN beta-1a in healthy clinical trial subjects

Allaire et al. BMC Research Notes 2013, 6:8 http://www.biomedcentral.com/1756-0500/6/8 RESEARCH ARTICLE Open Access Optimization of a high-throughput whole blood expression profiling methodology and its application to assess the pharmacodynamics of interferon (IFN) beta-1a or polyethylene glycol-conjugated IFN beta-1a in healthy clinical trial subjects Normand E Allaire1,2*, Steven E Bushnell1, Jadwiga Bienkowska1, Graham Brock1 and John Carulli1 Abstract Background: Clinical trials offer a unique opportunity to study human disease and response to therapy in a highly controlled setting. The application of high-throughput expression profiling to peripheral blood from clinical trial subjects could facilitate the identification of transcripts that function as prognostic or diagnostic markers of disease or treatment. The paramount issue for these methods is the ability to produce robust, reproducible, and timely mRNA expression profiles from peripheral blood. Single-stranded complementary DNA (sscDNA) targets derived from whole blood exhibit improved detection of transcripts and reduced variance as compared to their complementary RNA counterparts and therefore provide a better option for interrogation of peripheral blood on oligonucleotide arrays. High-throughput microarray technologies such as the high-throughput plate array platform offer several advantages compared with slide- or cartridge-based arrays; however, manufacturer’s protocols do not support the use of sscDNA targets. Results: We have developed a highly reproducible, high-through put, whole blood expression profiling methodology based on sscDNA and used it to analyze human brain reference RNA and universal human reference RNA samples to identify experimental conditions that most highly correlated with a gold standard quantitative polymerase chain reaction reference dataset. We then utilized the optimized method to analyze whole blood samples from healthy clinical trial subjects treated with different versions of interferon (IFN) beta-1a. Analysis of whole blood samples before and after treatment with intramuscular [IM] IFN beta-1a or polyethylene glycol-conjugated IFN (PEG-IFN) beta-1a under optimized experimental conditions demonstrated that PEG-IFN beta-1a induced a more sustained and prolonged pharmacodynamic response than unmodified IM IFN beta-1a. These results provide validation of the utility of this new methodology and suggest the potential therapeutic benefit of a sustained pharmacodynamic response to PEG-IFN beta-1a. Conclusions: This novel microarray methodology is ideally suited for utilization in large clinical studies to identify expressed transcripts for the elucidation of disease mechanisms of action and as prognostic, diagnostic, or toxicity markers. * Correspondence: 1 Biogen Idec Inc., Genetics and Genomics Group, 14 Cambridge Center, Cambridge, MA 02142, USA 2 Biogen Idec Inc., Translational Medicine Department, 14 Cambridge Center, Cambridge, MA 02142, USA © 2013 Allaire et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Allaire et al. BMC Research Notes 2013, 6:8 http://www.biomedcentral.com/1756-0500/6/8 Background The study of the blood transcriptome in the context of clinical pharmacogenomics has generated much interest in recent years [1,2]. The cellular and molecular components of peripheral blood exhibit dynamic responsiveness to physiological, environmental, or pathological stimuli and are in contact with nearly every tissue in the body, allowing for assessment of systemic responses to disease or treatment. As such, peripheral blood is a source of clinically accessible diagnostic, prognostic and pharmacodynamic (PD) markers [3,4]. This idea is supported by a growing body of research that describes the identification of expressed transcripts from human and animal peripheral blood that can function as indicators of disease, as prognostic markers of clinical outcome, of risk of toxicity, and as evidence of a therapy’s pharmacodynamic effects [5-8]. The successful use of gene expression microarrays in basic research studies has spawned great interest in the application of this technology to large clinical pharmacogenomics and population-based studies [9-11]. However, microarray cost, the complexity of sample processing and tracking, and practical limitations in sample throughput have restricted its utilization in clinical investigations [12,13]. Microarray manufacturers have responded to these needs with the recent development of higherthroughput solutions such as the high-throughput (HT) plate array or “array of arrays” [14]. This platform was made possible through reduction and optimization of probe content and advances in photonics, collectively enabling the miniaturization and assembly of 96 arrays into the spatial arrangement of a conventional microtiter plate. Our laboratory’s internal validation studies have confirmed that data from the HT plate array platform is highly concordant to that of industry standard cartridge arrays [15]. RNA is often amplified using T7 RNA polymerase-driven in vitro transcription (IVT) [16] to produce complementary RNA (cRNA) targets for hybridization to microarrays. However, the high concentration of hemoglobin transcripts in peripheral blood can induce a globin interference effect, effectively reducing a microarray’s detection sensitivity and increasing its signal variability [17]. Although effective methods have been developed to reduce globin interference [18-20], current methods of mitigation also induce variance in microarray results [21]. The challenges associated with utilizing cRNA targets from peripheral blood as probes for microarray investigations have led to the development of alternative methods of amplification and the use of single-stranded complementary DNA (sscDNA) targets from peripheral blood for microarray hybridization [22,23], effectively improving the sensitivity of microarray hybridizations for detecting peripheral blood transcripts. Results from our laboratory’s internal benchmarking experiments analyzing peripheral Page 2 of 10 blood samples have verified that sscDNA targets improve microarray sensitivity and decrease signal variance as compared with cRNA targets analyzed using globin blocking, degradation, and depletion methods (data not shown). In the current study, we have systematically optimized sscDNA/HT plate array target mass, hybridization parameters and washing parameters using 2 highly characterized test RNAs with the goal of developing a HT methodology for whole blood transcriptional profiling. Comparative analysis of optimization data against peerreviewed expression array [15] and quantitative polymerase chain reaction (qPCR) [24] datasets were u (...truncated)