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 Allaire,Steven E Bushnell,John Carulli,Graham Brock,Jadwiga Bienkowska

doi:10.1186/1756-0500-6-8

Abstract

BackgroundClinical 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.ResultsWe 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.ConclusionsThis 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.

Highlights

Clinical trials offer a unique opportunity to study human disease and response to therapy in a highly controlled setting
Relative variable effect To identify optimal hybridization conditions that both maximize detection of rare transcripts and minimize hybridization variance, labeled Single-stranded complementary DNA (sscDNA) generated in bulk from human brain reference RNA (HBRR) and universal human reference RNA (UHRR) were hybridized to HTHGU133A plate arrays using different masses under varying hybridization and washing conditions
We repeated calculations and analyses using R and Bioconductor computational tools as described by Gentleman [25], and to discover qualifiers that were differentially expressed between Human Brain Reference RNA (HBRR) and Universal Human Reference RNA (UHRR) for each condition tested, we applied the linear modeling approach (MANOVA) to fit gene expression levels according to the defined groups of samples and Bayesian posterior error analysis as implemented by Smyth [26]

Summary

Introduction

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. 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]

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