title: Reduction of systematic bias in transcriptome data from human peripheral blood mononuclear cells for transportation and biobanking.
abstract: Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. Therefore, to collect high-quality biobank samples for expression analysis, specimens must be transported under stable conditions. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from peripheral blood mononuclear cells (PBMCs) to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We randomly chose 7 samples from 11 samples individually, and systematic bias in expression levels was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA-Seq) experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. qRT-PCR experiments for 19 selected transcripts revealed systematic bias in the expression levels of five transcripts. RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, qRT-PCR and RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ohmomo
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan; Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hachiya
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shiwa
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Furukawa
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Ono
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Iwate, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Ito
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Iwate, Japan.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan; Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan; Division of Cardioangiology, Department of Internal Medicine and Memorial Heart Center, Iwate Medical University School of Medicine, Morioka, Iwate, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.affiliation: Department of Anatomy, Iwate Medical University School of Medicine, Shiwa-gun, Iwate, Japan; Deputy Executive Director, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Hitomi
authors.affiliation: Department of Neuroscience, Institute for Biomedical Sciences, Iwate Medical University, Shiwa-gun, Iwate, Japan; Executive Director, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sobue
authors.affiliation: Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shimizu
mesh_terms: D000328:Adult; D001771:Blood Banks; D001793:Blood Preservation; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D020871:RNA Stability; D017423:Sequence Analysis, RNA; D059467:Transcriptome
references.citation: J Clin Microbiol. 2000 Dec;38(12):4463-70
references.pmid: 11101581
references.citation: Eur Heart J. 2012 Nov;33(22):2796-803
references.pmid: 22930461
references.citation: BMC Genomics. 2009 Jan 05;10:2
references.pmid: 19123946
references.citation: PLoS One. 2012;7(11):e50721
references.pmid: 23209817
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: Stroke. 2013 Jun;44(6):1518-24
references.pmid: 23640824
references.citation: J Thromb Haemost. 2003 May;1(5):987-91
references.pmid: 12871366
references.citation: Bioinformatics. 2009 May 1;25(9):1105-11
references.pmid: 19289445
references.citation: Pharmacogenomics J. 2004;4(3):193-207
references.pmid: 15037859
references.citation: Clin Cancer Res. 2005 Feb 1;11(3):1181-9
references.pmid: 15709187
references.citation: Bioinformatics. 2009 Aug 15;25(16):2078-9
references.pmid: 19505943
references.citation: PLoS One. 2014 Jan 27;9(1):e87508
references.pmid: 24475299
references.citation: BMC Genomics. 2008 Oct 10;9:474
references.pmid: 18847473
references.citation: Am J Cardiol. 2012 Dec 15;110(12):1856-60
references.pmid: 22999072
references.citation: Nat Biotechnol. 2013 Jan;31(1):46-53
references.pmid: 23222703
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Nat Biotechnol. 2010 May;28(5):511-5
references.pmid: 20436464
references.citation: Clin Chem. 2002 Nov;48(11):1883-90
references.pmid: 12406972
references.citation: J Exp Med. 1983 Aug 1;158(2):393-412
references.pmid: 6411852
references.citation: Int J Epidemiol. 2008 Apr;37(2):234-44
references.pmid: 18381398
references.citation: Epidemiology. 2002 May;13(3):246-54
references.pmid: 11964924
references.citation: PLoS One. 2011 Feb 10;6(2):e17014
references.pmid: 21347333
references.citation: Clin Chem. 2008 Feb;54(2):396-405
references.pmid: 18089658
references.citation: Biopreserv Biobank. 2012 Jun;10(3):266-75
references.pmid: 24835065
references.citation: Genomics. 2006 May;87(5):653-64
references.pmid: 16387473