1921
Volume 98, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645

Abstract

Abstract.

Obtaining RNA from clinical samples collected in resource-limited settings can be costly and challenging. The goals of this study were to 1) optimize messenger RNA extraction from dried blood spots (DBS) and 2) determine how transcriptomes generated from DBS RNA compared with RNA isolated from blood collected in Tempus tubes. We studied paired samples collected from eight adults in rural Tanzania. Venous blood was collected on Whatman 903 Protein Saver cards and in tubes with RNA preservation solution. Our optimal DBS RNA extraction used 8 × 3-mm DBS punches as the starting material, bead beater disruption at maximum speed for 60 seconds, extraction with Illustra RNAspin Mini RNA Isolation kit, and purification with Zymo RNA Concentrator kit. Spearman correlations of normalized gene counts in DBS versus whole blood ranged from 0.887 to 0.941. Bland–Altman plots did not show a trend toward over- or under-counting at any gene size. We report a method to obtain sufficient RNA from DBS to generate a transcriptome. The DBS transcriptome gene counts correlated well with whole blood transcriptome gene counts. Dried blood spots for transcriptome studies could be an option when field conditions preclude appropriate collection, storage, or transport of whole blood for RNA studies.

Loading

Article metrics loading...

The graphs shown below represent data from March 2017
/content/journals/10.4269/ajtmh.17-0653
2018-03-05
2019-11-19
Loading full text...

Full text loading...

/deliver/fulltext/14761645/98/5/tpmd170653.html?itemId=/content/journals/10.4269/ajtmh.17-0653&mimeType=html&fmt=ahah

References

  1. Hedegaard J, 2014. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One 9: e98187. [Google Scholar]
  2. Schuierer S, Carbone W, Knehr J, Petitjean V, Fernandez A, Sultan M, Roma G, , 2017. A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples. BMC Genomics 18: 442. [Google Scholar]
  3. Patton JC, Akkers E, Coovadia AH, Meyers TM, Stevens WS, Sherman GG, , 2007. Evaluation of dried whole blood spots obtained by heel or finger stick as an alternative to venous blood for diagnosis of human immunodeficiency virus type 1 infection in vertically exposed infants in the routine diagnostic laboratory. Clin Vaccine Immunol 14: 201203. [Google Scholar]
  4. Gauffin F, Nordgren A, Barbany G, Gustafsson B, Karlsson H, , 2009. Quantitation of RNA decay in dried blood spots during 20 years of storage. Clin Chem Lab Med 47: 14671469. [Google Scholar]
  5. Ponnusamy V, Kapellou O, Yip E, Evanson J, Wong LF, Michael-Titus A, Yip PK, Shah DK, , 2016. A study of microRNAs from dried blood spots in newborns after perinatal asphyxia: a simple and feasible biosampling method. Pediatr Res 79: 799805. [Google Scholar]
  6. Maeno Y, Nakazawa S, Nagashima S, Sasaki J, Higo KM, Taniguchi K, , 2003. Utility of the dried blood on filter paper as a source of cytokine mRNA for the analysis of immunoreactions in Plasmodium yoelii infection. Acta Trop 87: 295300. [Google Scholar]
  7. Haak PT, Busik JV, Kort EJ, Tikhonenko M, Paneth N, Resau JH, , 2009. Archived unfrozen neonatal blood spots are amenable to quantitative gene expression analysis. Neonatology 95: 210216. [Google Scholar]
  8. Khoo SK, Dykema K, Vadlapatla NM, LaHaie D, Valle S, Satterthwaite D, Ramirez SA, Carruthers JA, Haak PT, Resau JH, , 2011. Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays. Pathol Int 61: 16. [Google Scholar]
  9. Ho NT, 2013. Gene expression in archived newborn blood spots distinguishes infants who will later develop cerebral palsy from matched controls. Pediatr Res 73: 450456. [Google Scholar]
  10. Grauholm J, Khoo SK, Nickolov RZ, Poulsen JB, Baekvad-Hansen M, Hansen CS, Hougaard DM, Hollegaard MV, , 2015. Gene expression profiling of archived dried blood spot samples from the Danish Neonatal Screening Biobank. Mol Genet Metab 116: 119124. [Google Scholar]
  11. McDade TW, Ross K, Fried R, Arevalo JM, Ma J, Miller GE, Cole SW, , 2016. Genome-wide profiling of RNA from dried blood spots: convergence with bioinformatic results derived from whole venous blood and peripheral blood mononuclear cells. Biodemography Soc Biol 62: 182197. [Google Scholar]
  12. Bybjerg-Grauholm J, Hagen CM, Khoo SK, Johannesen ML, Hansen CS, Baekvad-Hansen M, Christiansen M, Hougaard DM, Hollegaard MV, , 2017. RNA sequencing of archived neonatal dried blood spots. Mol Genet Metab Rep 10: 3337. [Google Scholar]
  13. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL, , 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14: R36. [Google Scholar]
  14. Anders S, Pyl PT, Huber W, , 2015. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31: 166169. [Google Scholar]
  15. Love MI, Huber W, Anders S, , 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550. [Google Scholar]
  16. Robinson MD, Oshlack A, , 2010. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11: R25. [Google Scholar]
  17. Altman DG, Bland JM, , 1983. Measurement in medicine—the analysis of method comparison studies. Statistician 32: 307317. [Google Scholar]
  18. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T, , 2006. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7: 3. [Google Scholar]
  19. Webster AF, Zumbo P, Fostel J, Gandara J, Hester SD, Recio L, Williams A, Wood CE, Yauk CL, Mason CE, , 2015. Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded tissues. Toxicol Sci 148: 460472. [Google Scholar]
  20. Fajardo E, Metcalf CA, Chaillet P, Aleixo L, Pannus P, Panunzi I, Triviño L, Ellman T, Likaka A, Mwenda R, , 2014. Prospective evaluation of diagnostic accuracy of dried blood spots from finger prick samples for determination of HIV-1 load with the NucliSENS Easy-Q HIV-1 version 2.0 assay in Malawi. J Clin Microbiol 52: 13431351. [Google Scholar]
http://instance.metastore.ingenta.com/content/journals/10.4269/ajtmh.17-0653
Loading
/content/journals/10.4269/ajtmh.17-0653
Loading

Data & Media loading...

Supplemental Figures and Table

  • Received : 17 Aug 2017
  • Accepted : 14 Jan 2018
  • Published online : 05 Mar 2018

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error