Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods

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.

利用血液样本开展基因表达谱分析（gene expression profiling），是临床研究中用于生物标志物（biomarker）发现的重要工具。不同的全血RNA采集与处理方法存在显著差异，可能会混淆不同研究间的结果比对。本研究的核心目标为对比血液保存方式、提取方法以及血液自身组分对基因表达谱分析的影响。本研究从5名健康志愿者体内采集三份重复的全血与外周血单个核细胞（peripheral blood mononuclear cell, PBMC）样本。全血样本分别采集于RNAgard®、PAXgene®血液RNA采集管，以及含有乙二胺四乙酸二钾（K2EDTA）、柠檬酸钠葡萄糖溶液A（ACD-A）等抗凝剂的采血管中。PBMC则分别采用柠檬酸钠细胞制备管（CPT™）、FICOLL™、磁珠分离法以及LeukoLOCK™方法进行分离。采血完成后，LeukoLOCK™采血管、K2EDTA采血管与ACD-A采血管需在低温条件下隔夜运输；其余采集方式的样本则在完成预处理或不进行预处理的情况下立即冻存。本研究共采用10种不同实验条件，依托多款广泛应用的RNA分离方法，从全血与PBMC样本中提取RNA。采用RNA完整性数（RNA Integrity Number, RIN）评估RNA质量，结果显示，若在RNA提取前使用TRIzol®试剂对血液进行稳定化处理，所有PBMC分离流程的RIN值均为最高。初步数据分析显示，与经冻存稳定化处理的血液相比，在4℃条件下保存并隔夜运输的人体血液样本，其RNA完整性数检测结果同样优异。对供体内部、方法学重复样本间的比对分析显示，样本的信噪比特征无法仅通过RIN值体现。基于前1000个经错误发现率（false discovery rate, FDR）校正的差异表达基因（differentially expressed genes, DEGs）进行通路分析，结果显示：全血样本的冻存与低温运输条件对基因表达模式存在显著影响。然而，针对经低温或冻存运输保存的PBMC样本（采用RNAlater®稳定的LeukoLOCK™方法）的前1000个经FDR校正的DEGs进行分析，并未发现存在显著受影响的通路。本研究结果揭示了RNA分离、各类保存、操作及处理方法如何影响血液样本的RNA质量与表观基因表达，为相关领域提供了全新视角。研究人员需谨慎考量，以规避下游处理流程引入的偏倚。进一步明确不同采集方法的特性影响，将有助于深入探究人类样本类型中基因表达异质性的作用机制。