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


Autoři: Aarti Gautam aff001;  Duncan Donohue aff001;  Allison Hoke aff001;  Stacy Ann Miller aff001;  Seshamalini Srinivasan aff001;  Bintu Sowe aff001;  Leanne Detwiler aff001;  Jesse Lynch aff001;  Michael Levangie aff001;  Rasha Hammamieh aff001;  Marti Jett aff001
Působiště autorů: US Army Center for Environmental Health Research, Fort Detrick, MD, United States of America aff001;  The Geneva Foundation, US Army Center for Environmental Health Research, Fort Detrick, MD, United States of America aff002;  Oak Ridge Institute for Science and Education, Fort Detrick, US Army Center for Environmental Health Research, Fort Detrick, MD, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225137

Souhrn

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.

Klíčová slova:

Blood – Gene expression – Lymphocytes – RNA extraction – RNA isolation – Specimen storage – T cells – White blood cells


Zdroje

1. Bushel PR, Heinloth AN, Li J, Huang L, Chou JW, Boorman GA, et al. Blood gene expression signatures predict exposure levels. Proceedings of the National Academy of Sciences. 2007;104(46):18211–6. doi: 10.1073/pnas.0706987104 17984051

2. Hofmann T, Klenow S, Borowicki A, Gill CIR, Pool-Zobel BL, Glei M. Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations. Genes & Nutrition. 2010;5(4):309–19. doi: 10.1007/s12263-010-0170-1 PMC2989368. 21189867

3. Gliddon HD, Herberg JA, Levin M, Kaforou M. Genome‐wide host RNA signatures of infectious diseases: discovery and clinical translation. Immunology. 2018;153(2):171–8. doi: 10.1111/imm.12841 PMC5765383. 28921535

4. Asare AL, Kolchinsky SA, Gao Z, Wang R, Raddassi K, Bourcier K, et al. Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation. BMC genomics. 2008;9:474. doi: 10.1186/1471-2164-9-474 18847473; PubMed Central PMCID: PMC2573897.

5. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Moser K, Ortmann WA, et al. Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation. Genes and immunity. 2004;5(5):347–53. doi: 10.1038/sj.gene.6364098 15175644.

6. Carrol ED, Salway F, Pepper SD, Saunders E, Mankhambo LA, Ollier WE, et al. Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis. BMC immunology. 2007;8:20. doi: 10.1186/1471-2172-8-20 17850649; PubMed Central PMCID: PMC2031894.

7. Corkum CP, Ings DP, Burgess C, Karwowska S, Kroll W, Michalak TI. Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT) and standard density gradient. BMC immunology. 2015;16:48. doi: 10.1186/s12865-015-0113-0 26307036; PubMed Central PMCID: PMC4549105.

8. Debey-Pascher S, Hofmann A, Kreusch F, Schuler G, Schuler-Thurner B, Schultze JL, et al. RNA-Stabilized Whole Blood Samples but Not Peripheral Blood Mononuclear Cells Can Be Stored for Prolonged Time Periods Prior to Transcriptome Analysis. The Journal of molecular diagnostics: JMD. 2011;13(4):452–60. doi: 10.1016/j.jmoldx.2011.03.006 PMC3123794. 21704280

9. Franken C, Remy S, Lambrechts N, Hollanders K, Den Hond E, Schoeters G. Peripheral blood collection: the first step towards gene expression profiling. Biomarkers: biochemical indicators of exposure, response, and susceptibility to chemicals. 2016;21(5):458–65. doi: 10.3109/1354750X.2016.1153721 26984061.

10. Malentacchi F, Pazzagli M, Simi L, Orlando C, Wyrich R, Gunther K, et al. SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. PloS one. 2014;9(11):e112293. doi: 10.1371/journal.pone.0112293 25384019; PubMed Central PMCID: PMC4226503.

11. Pazzagli M, Malentacchi F, Simi L, Orlando C, Wyrich R, Gunther K, et al. SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. Methods. 2013;59(1):20–31. doi: 10.1016/j.ymeth.2012.10.007 23110812.

12. Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C, Schram J, et al. Stabilization of mRNA expression in whole blood samples. Clinical chemistry. 2002;48(11):1883–90. 12406972.

13. Robison EH, Mondala TS, Williams AR, Head SR, Salomon DR, Kurian SM. Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study. BMC genomics. 2009;10:617. doi: 10.1186/1471-2164-10-617 20017944; PubMed Central PMCID: PMC2811129.

14. Shabihkhani M, Lucey GM, Wei B, Mareninov S, Lou JJ, Vinters HV, et al. The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings. Clinical biochemistry. 2014;47(4–5):258–66. doi: 10.1016/j.clinbiochem.2014.01.002 24424103; PubMed Central PMCID: PMC3982909.

15. Shou J, Dotson C, Qian HR, Tao W, Lin C, Lawrence F, et al. Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray. Biomarkers: biochemical indicators of exposure, response, and susceptibility to chemicals. 2005;10(4):310–20. doi: 10.1080/13547500500218583 16191486.

16. Thach DC, Lin B, Walter E, Kruzelock R, Rowley RK, Tibbetts C, et al. Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays. Journal of immunological methods. 2003;283(1–2):269–79. doi: 10.1016/j.jim.2003.10.004 14659918.

17. Jiang Z, Uboh CE, Chen J, Soma LR. Isolation of RNA from equine peripheral blood cells: comparison of methods. SpringerPlus. 2013;2(1):478. doi: 10.1186/2193-1801-2-478 PMC3797321. 24133642

18. Schwochow D, Serieys LEK, Wayne RK, Thalmann O. Efficient recovery of whole blood RNA—a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species. BMC Biotechnology. 2012;12:33–. doi: 10.1186/1472-6750-12-33 PMC3406948. 22738215

19. Rinchai D, Anguiano E, Nguyen P, Chaussabel D. Finger stick blood collection for gene expression profiling and storage of tempus blood RNA tubes. F1000Research. 2016;5:1385. doi: 10.12688/f1000research.8841.2 28357036; PubMed Central PMCID: PMC5357033.

20. Menke A, Rex-Haffner M, Klengel T, Binder EB, Mehta D. Peripheral blood gene expression: it all boils down to the RNA collection tubes. BMC research notes. 2012;5:1. doi: 10.1186/1756-0500-5-1 22214347; PubMed Central PMCID: PMC3280191.

21. Joehanes R, Johnson AD, Barb JJ, Raghavachari N, Liu P, Woodhouse KA, et al. Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study. Physiological genomics. 2012;44(1):59–75. doi: 10.1152/physiolgenomics.00130.2011 22045913; PubMed Central PMCID: PMC3289123.

22. Bondar G, Cadeiras M, Wisniewski N, Maque J, Chittoor J, Chang E, et al. Comparison of Whole Blood and Peripheral Blood Mononuclear Cell Gene Expression for Evaluation of the Perioperative Inflammatory Response in Patients with Advanced Heart Failure. PloS one. 2014;9(12):e115097. doi: 10.1371/journal.pone.0115097 PMC4269402. 25517110

23. Min JL, Barrett A, Watts T, Pettersson FH, Lockstone HE, Lindgren CM, et al. Variability of gene expression profiles in human blood and lymphoblastoid cell lines. BMC genomics. 2010;11(1):96. doi: 10.1186/1471-2164-11-96 20141636

24. Yang J, Diaz N, Adelsberger J, Zhou X, Stevens R, Rupert A, et al. The effects of storage temperature on PBMC gene expression. BMC immunology. 2016;17(1):6. doi: 10.1186/s12865-016-0144-1 26979060

25. Mallone R, Mannering SI, Brooks-Worrell BM, Durinovic-Belló I, Cilio CM, Wong FS, et al. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clinical and Experimental Immunology. 2011;163(1):33–49. doi: 10.1111/j.1365-2249.2010.04272.x PMC3010910. 20939860

26. Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA, et al. Individuality and variation in gene expression patterns in human blood. Proceedings of the National Academy of Sciences. 2003;100(4):1896–901. doi: 10.1073/pnas.252784499 12578971

27. Dagur PK, McCoy JP. Collection, Storage, and Preparation of Human Blood Cells. Current protocols in cytometry / editorial board, J Paul Robinson, managing editor [et al]. 2015;73:5.1.-5.1.16. doi: 10.1002/0471142956.cy0501s73 PMC4524540. 26132177

28. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Research. 2015;43(7):e47–e. doi: 10.1093/nar/gkv007 PMC4402510. 25605792

29. Sammon JW. A Nonlinear Mapping for Data Structure Analysis. IEEE Trans Comput. 1969;18(5):401–9. doi: 10.1109/t-c.1969.222678

30. McShine RL, Das PC, Sibinga CTS, Brozovic B. Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume. Clinical & Laboratory Haematology. 1990;12(3):277–85. doi: 10.1111/j.1365-2257.1990.tb00038.x 2125542

31. Donohue DE, Gautam A, Miller S-A, Srinivasan S, Abu-Amara D, Campbell R, et al. Gene expression profiling of whole blood: A comparative assessment of RNA-stabilizing collection methods. PloS one. 2019;14(10):e0223065–e. doi: 10.1371/journal.pone.0223065 31600258.

32. Weckle A, Aiello AE, Uddin M, Galea S, Coulborn RM, Soliven R, et al. Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting. Journal of visualized experiments: JoVE. 2015;(105):52227. doi: 10.3791/52227 PMC4692771. 26649992

33. Yu L, Warner P, Warner B, Recktenwald D, Yamanishi D, Guia A, et al. Whole blood leukocytes isolation with microfabricated filter for cell analysis. Cytometry Part A. 2011;79A(12):1009–15. doi: 10.1002/cyto.a.21114 22110022

34. Bayatti N, Cooper-Knock J, Bury JJ, Wyles M, Heath PR, Kirby J, et al. Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis. PloS one. 2014;9(1):e87508. doi: 10.1371/journal.pone.0087508 PMC3903649. 24475299

35. Kang J E., Hwang S, H. Lee J, Park do Y, H. Kim H. Effects of RBC removal and TRIzol of peripheral blood samples on RNA stability2011. 1883–5 p.

36. Kang J-E, Hwang S-H, Lee JH, Park DY, Kim H-H. Effects of RBC removal and TRIzol of peripheral blood samples on RNA stability. Clinica Chimica Acta. 2011;412(19):1883–5. https://doi.org/10.1016/j.cca.2011.06.016.

37. Lee J-M, Kang JS. Changes of hematological references depends on storage period and temperature conditions in rats and dogs. Laboratory Animal Research. 2016;32(4):241–8. doi: 10.5625/lar.2016.32.4.241 PMC5206231. 28053618

38. Francesca M, Sara P, Ralf W, Paolo V, Chiara C, Mario P, et al. Effects of Transport and Storage Conditions on Gene Expression in Blood Samples. Biopreservation and Biobanking. 2016;14(2):122–8. doi: 10.1089/bio.2015.0037 26886447.

39. Uyuklu M, Cengiz MS, Ulker P, Hever T, Tripette J, Connes P, et al. Effects of storage duration and temperature of human blood on red cell deformability and aggregation2009. 269–78 p.

40. Huang L-H, Lin P-H, Tsai K-W, Wang L-J, Huang Y-H, Kuo H-C, et al. The effects of storage temperature and duration of blood samples on DNA and RNA qualities. PloS one. 2017;12(9):e0184692. doi: 10.1371/journal.pone.0184692 28926588

41. Djaldetti M, Bessler H. High temperature affects the phagocytic activity of human peripheral blood mononuclear cells. Scandinavian Journal of Clinical and Laboratory Investigation. 2015;75(6):482–6. doi: 10.3109/00365513.2015.1052550 26067609


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