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- INTRODUCTION
- MATERIALS AND METHODS
- Sample collection.
- Ethics.
- Optimization of RNA quality and yield from DBSsamples.
- RNA extraction method.
- Sample disruption.
- Optimization of homogenizer settings and input DBS.
- Preparation of RNA for transcriptome studies.
- RNA quality assessment.
- RNA sample library preparation.
- Sequencing with Illumina HiSeq 4000.
- Statistical methods.
- RESULTS
- DISCUSSION
- Supplementary Material
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Hedegaard J et al. 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.
-
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.
-
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: 201–203.
-
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: 1467–1469.
-
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: 799–805.
-
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: 295–300.
-
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: 210–216.
-
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: 1–6.
-
9.
Ho NT et al. 2013. Gene expression in archived newborn blood spots distinguishes infants who will later develop cerebral palsy from matched controls. Pediatr Res 73: 450–456.
-
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: 119–124.
-
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: 182–197.
-
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: 33–37.
-
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.
-
14.
Anders S, Pyl PT, Huber W, 2015. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31: 166–169.
-
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.
-
16.
Robinson MD, Oshlack A, 2010. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11: R25.
-
17.
Altman DG, Bland JM, 1983. Measurement in medicine—the analysis of method comparison studies. Statistician 32: 307–317.
-
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.
-
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: 460–472.
-
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: 1343–1351.
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| Full Text Views | 1335 | 27 | 0 |
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Dried Blood Spot RNA Transcriptomes Correlate with Transcriptomes Derived from Whole Blood RNA
Mary J. Reust
Mary J. Reust
Department of Medicine, Weill Cornell Medicine, New York, New York;
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Myung Hee Lee
Myung Hee Lee
Department of Medicine, Weill Cornell Medicine, New York, New York;
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Jenny Xiang
Jenny Xiang
Genomics Resources Core Facility, Weill Cornell Medicine, New York, New York
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Wei Zhang
Wei Zhang
Genomics Resources Core Facility, Weill Cornell Medicine, New York, New York
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Dong Xu
Dong Xu
Genomics Resources Core Facility, Weill Cornell Medicine, New York, New York
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Tatiana Batson
Tatiana Batson
Genomics Resources Core Facility, Weill Cornell Medicine, New York, New York
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Tuo Zhang
Tuo Zhang
Genomics Resources Core Facility, Weill Cornell Medicine, New York, New York
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Jennifer A. Downs
Jennifer A. Downs
Department of Medicine, Weill Cornell Medicine, New York, New York;
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Kathryn M. Dupnik
Kathryn M. Dupnik
Department of Medicine, Weill Cornell Medicine, New York, New York;
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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.
- Supplemental Materials (PDF 4154 KB)
Author Notes
Financial support: K. M. D. was supported by UL1-TR000457-06. K23-AI110238 provided project funding and support for M. J. R. and J. A. D. were supported in part by the Kellen Junior Faculty Fellowship at Weill Cornell Medicine. Funding agencies had no role in study design, data collection, interpretation of results, or the decision to publish.
Authors’ addresses: Mary J. Reust, Myung Hee Lee, Jennifer A. Downs, and Kathryn M. Dupnik, Department of Medicine, Weill Cornell Medicine, New York, NY, E-mails: mar9227@med.cornell.edu, myl2003@med.cornell.edu, jna2002@med.cornell.edu, and kad9040@med.cornell.edu. Jenny Xiang, Wei Zhang, Dong Xu, Tatiana Batson, and Tuo Zhang, Genomics Resources Core Facility, Weill Cornell Medicine, New York, NY, E-mails: jzx2002@med.cornell.edu, wez2009@med.cornell.edu, xud2001@med.cornell.edu, tab2022@med.cornell.edu, and taz2008@med.cornell.edu.
- INTRODUCTION
- MATERIALS AND METHODS
- Sample collection.
- Ethics.
- Optimization of RNA quality and yield from DBSsamples.
- RNA extraction method.
- Sample disruption.
- Optimization of homogenizer settings and input DBS.
- Preparation of RNA for transcriptome studies.
- RNA quality assessment.
- RNA sample library preparation.
- Sequencing with Illumina HiSeq 4000.
- Statistical methods.
- RESULTS
- DISCUSSION
- Supplementary Material
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Hedegaard J et al. 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.
-
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.
-
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: 201–203.
-
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: 1467–1469.
-
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: 799–805.
-
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: 295–300.
-
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: 210–216.
-
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: 1–6.
-
9.
Ho NT et al. 2013. Gene expression in archived newborn blood spots distinguishes infants who will later develop cerebral palsy from matched controls. Pediatr Res 73: 450–456.
-
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: 119–124.
-
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: 182–197.
-
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: 33–37.
-
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.
-
14.
Anders S, Pyl PT, Huber W, 2015. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31: 166–169.
-
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.
-
16.
Robinson MD, Oshlack A, 2010. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11: R25.
-
17.
Altman DG, Bland JM, 1983. Measurement in medicine—the analysis of method comparison studies. Statistician 32: 307–317.
-
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.
-
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: 460–472.
-
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: 1343–1351.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1247 | 911 | 320 |
| Full Text Views | 1335 | 27 | 0 |
| PDF Downloads | 736 | 24 | 0 |