Dataset Information

Peripheral blood gene expression: it all boils down to the RNA collection tubes.

ABSTRACT:

Background

Gene expression profiling from peripheral blood 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 study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgene™ and Tempus™ tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals.

Results

RNA quality, yield and numbers of detected transcripts from the two RNA collection systems was comparable, with no significant differences between the tube types. Globin reduction resulted in a significant increase in present call rates (p = 8.17 × 10-5 and p = 1.95 × 10-3 in PAXgene™ and Tempus™ tubes respectively) and significant decrease in gene expression variance in both RNA collection tubes (p = 0.0025 and p = 0.041 in PAXgene™ and Tempus™ tubes respectively). Comparisons of glucocorticoid receptor-stimulated gene expression profiles between the two collection tube systems revealed an overlap of only 17 to 54%, depending on the stringency level of the statistical thresholds. This overlap increased by 1-8% when the RNA samples were processed to remove the globin mRNA.

Conclusion

RNA obtained from PAXgene™ and Tempus™ tubes was comparable in terms of quality and yield, however, detectable gene expression changes after glucocorticoid receptor stimulation were distinct, with an overlap of only up to 46% between the two collection systems. This overlap increased to 54% when the samples were depleted of globin mRNA and drastically reduced to 17-18% when only gene expression differences with a fold change greater than 2.0 were assessed. These results indicate that gene expression profiles obtained from PAXgene™ and Tempus™ differ drastically and should not be analyzed together. These data suggest that researchers must exert caution while interpreting expression profiles obtained through different RNA collection tubes.

SUBMITTER: Menke A

PROVIDER: S-EPMC3280191 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Peripheral blood gene expression: it all boils down to the RNA collection tubes.

Menke Andreas A Rex-Haffner Monika M Klengel Torsten T Binder Elisabeth B EB Mehta Divya D Mehta Divya D

BMC research notes 20120104

<h4>Background</h4>Gene expression profiling from peripheral blood 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 study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgene™ and Tempus™ tubes. Comparisons of present call rat ...[more]

PMID: 22214347

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Project description: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). CONCLUSION: 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.

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.

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Project description:Several previous studies have attempted to investigate the regulatory mechanisms underlying gene expression in ankylosing spondylitis (AS). However, the specific molecular pathways underlying this condition remain unclear. Previous research used next-generation RNA sequencing to identify a series of differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) when compared between patients with AS and healthy controls, thus implying that these DEGs may be related to AS. Furthermore, by screening these DEGS, it may be possible to facilitate clinical diagnosis and optimize treatment strategies. In order to test this hypothesis, we recruited 15 patients with AS and 15 healthy controls. We randomly selected five subjects from each group of patients for RNA sequencing analysis. Sequence reads were generated by an Illumina HiSeq2500 platform and mapped on to the human reference genome using HISAT2. We successfully identified 973 significant DEGs (p < 0.05) in PBMCs. When compared with controls, 644 of these genes were upregulated (with a fold?change?(FC) > 2) in AS patients and 329 were downregulated (FC < 0.5). Our analysis identified numerous genes related to immune response. Gene Ontology (GO) analysis indicated that these DEGs were significantly related to the positive regulation of epidermal growth factor-activated receptor activity, the positive regulation of the ERBB (erb-b2 receptor tyrosine kinase) signaling pathway, the differentiation of trophoblast giant cells, oxygen transport, immune-related pathways, and inflammation-related pathways. The DEGs were also closely related to the TNF and NF-?B signaling pathways. Six DEGs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Receiver operating characteristic (ROC) curve analysis indicated that IL6 may represent a useful biomarker for diagnosing AS. The development of new biomarkers may help us to elucidate the specific mechanisms involved in the development and progression of AS.

Dataset Information

Peripheral blood gene expression: it all boils down to the RNA collection tubes.

Background

Results

Conclusion

Publications

Peripheral blood gene expression: it all boils down to the RNA collection tubes.

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