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Series GSE113395 Query DataSets for GSE113395
Status Public on Oct 31, 2019
Title Investigating Gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple methods
Organism Homo sapiens
Experiment type Expression profiling by array
Summary 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.
 
Overall design The objective of this study was to investigate how blood storage, extraction methodologies, and the blood component itself may influence what genes appear to be expressed in downstream applications. Blood was collected in triplicate from 5 donors into five different collection tubes. Few of the tubes were frozen post collection, while others were stored at 4°C overnight. The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) employing several widely utilized RNA isolation methods. After extraction, the RNA was quantified using a spectrophotometer and RNA quality was assessed by the RNA Integrity Number (RIN) value. The RNA was then profiled using Agilent Human Gene Expression microarrays to investigate the different expression levels for the experimental conditions. The RNA quality was assessed by RIN value and we found out 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 of blood collection methods within and across donor 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.
[Collection protocol] Blood was collected and preserved in five tubes, namely, PAXgene™ RNA tubes, RNAgard® Blood Tubes, BD Vacutainer™ tubes with ACD-A and K2EDTA and CPT (LeukoLOCK) tubes from five healthy volunteers. In parallel, PBMCs were isolated from EDTA, CPT and ACD-A tubes following the manufacturer’s instructions.
 
Contributor(s) Gautam A, Hammamieh R, Jett M
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Submission date Apr 19, 2018
Last update date Nov 02, 2019
Contact name Aarti Gautam
E-mail(s) aarti.gautam.civ@health.mil
Phone 301-619-7683
Organization name WRAIR
Department Medical Readiness Systems Biology
Street address 503 Robert Grant Avenue
City Silver Spring
State/province MD
ZIP/Postal code 20910
Country USA
 
Platforms (1)
GPL17077 Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray 039381 (Probe Name version)
Samples (150)
GSM3104920 13872_ACD-A _ACK lysing replicate 1
GSM3104921 13872_ACD-A _ACK lysing replicate 2
GSM3104922 13872_ACD-A _ACK lysing replicate 3
Relations
BioProject PRJNA451015

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE113395_RAW.tar 892.4 Mb (http)(custom) TAR (of TXT)
Processed data included within Sample table

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