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Genome-wide analysis of the effect of long-term freezing of PAXgene Blood RNA tubes

ABSTRACT: Analysis of long-term freezing on the stability of transcriptome profiles in PAXgene stabilized whole blood samples. In the present study it was tested if long-term freezing of PAXgene RNA tubes (up to one year) has an influence on the transcriptome profile of peripheral whole blood samples. Results indicated that gene expression profiles of whole blood samples stabilized with PAXgene RNA tubes remain stable for at least 1 year.

ORGANISM(S): Homo sapiens

PROVIDER: GSE24757 | GEO | 2011/07/22

SECONDARY ACCESSION(S): PRJNA133395

REPOSITORIES: GEO

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Project description:Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results: We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion: RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. Keywords: Whole Blood, Protocol Variation, Expression Profiling, Microarray, globin, PAXGene, PNA

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 study was to compare how blood storage, extraction methodologies, and the blood component itself may influence gene expression profiling where samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAGard™, PAXgene™ as well as collection tubes with anticoagulant such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and acid citrate dextrose solution A (ACD-A). Peripheral blood mononuclear cells (PBMCs) were separated using sodium citrate cell preparation tubes (CPT), FICOLL™, immunomagnetic and Leukolock™ methods. The Leukolock™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and rest of the methods were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) with a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality as assessed by RNA integrity Number (RIN) showed that all PBMC procedures had the highest RIN values when processed and stabilized in TRIzol before RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well 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. Our results provide some new insights into RNA isolation from blood samples, how the RNA isolation quality and apparent gene expression is influenced by the choice of storage, handling, and methodologies, and how careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research into understanding the effect of gene expression on variability in human sample types.

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Genome-wide analysis of the effect of long-term freezing of PAXgene Blood RNA tubes

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