Dataset Information

A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

ABSTRACT:

Background

Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study.

Methods and findings

We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal.

Conclusion

We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study.

SUBMITTER: Tian Z

PROVIDER: S-EPMC2668177 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Publications

A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

Tian Ze Z Palmer Nathan N Schmid Patrick P Yao Hui H Galdzicki Michal M Berger Bonnie B Wu Erxi E Kohane Isaac S IS

PloS one 20090417 4

<h4>Background</h4>Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study.<h4>Methods and findings</h4>We applied peripheral whole blood samples with globin redu ...[more]

PMID: 19381341

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Project description: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.

Project description:Gene expression profiling using blood samples is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the current study is to compare how blood storage, extraction methodologies, and the blood components themselves may influence gene expression profiling. Whole blood and peripheral blood mononuclear cell (PBMC) samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAgard® and PAXgene® Blood RNA Tubes, as well as in collection tubes with anticoagulants such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and Acid Citrate Dextrose Solution A (ACD-A). PBMCs were separated using sodium citrate Cell Preparation Tubes (CPT™), FICOLL™, magnetic separation, and the LeukoLOCK™ methods. After blood collection, the LeukoLOCK™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and samples from the rest of the collection were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and PBMCs using a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality was assessed by RNA Integrity Number (RIN), which showed that all PBMC procedures had the highest RIN values when blood was stabilized in TRIzol® Reagent before RNA extraction. Initial data analysis showed that human blood stored and shipped at 4°C overnight performed equally well when checked for quality using RNA integrity number when compared to frozen stabilized blood. Comparisons within and across donor/method replicates showed signal-to-noise patterns which were not captured by RIN value alone. Pathway analysis using the top 1000 false discovery rate (FDR) corrected differentially expressed genes (DEGs) showed frozen vs. cold shipping conditions greatly impacted gene expression patterns in whole blood. However, the top 1000 FDR corrected DEGs from PBMCs preserved after frozen vs. cold shipping conditions (LeukoLOCK™ preserved in RNAlater®) revealed no significantly affected pathways. Our results provide novel insight into how RNA isolation, various storage, handling, and processing methodologies can influence RNA quality and apparent gene expression using blood samples. Careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research in understanding the effect of gene expression variability in human sample types.

Dataset Information

A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

Background

Methods and findings

Conclusion

Publications

A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

Similar Datasets

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