All Title Author
Keywords Abstract

Evaluation of Sex-Specific Gene Expression in Archived Dried Blood Spots (DBS)

DOI: 10.3390/ijms13089599

Keywords: archived dried blood spots (DBS), sex-specific, gene expression, molecular genetic profiling, microarray

Full-Text   Cite this paper   Add to My Lib


Screening newborns for treatable serious conditions is mandated in all US states and many other countries. After screening, Guthrie cards with residual blood (whole spots or portions of spots) are typically stored at ambient temperature in many facilities. The potential of archived dried blood spots (DBS) for at-birth molecular studies in epidemiological and clinical research is substantial. However, it is also challenging as analytes from DBS may be degraded due to preparation and storage conditions. We previously reported an improved assay for obtaining global RNA gene expression from blood spots. Here, we evaluated sex-specific gene expression and its preservation in DBS using oligonucleotide microarray technology. We found X inactivation-specific transcript ( XIST), lysine-specific demethylase 5D ( KDM5D) (also known as selected cDNA on Y, homolog of mouse ( SMCY)), uncharacterized LOC729444 ( LOC729444), and testis-specific transcript, Y-linked 21 ( TTTY21) to be differentially-expressed by sex of the newborn. Our finding that trait-specific RNA gene expression is preserved in unfrozen DBS, demonstrates the technical feasibility of performing molecular genetic profiling using such samples. With millions of DBS potentially available for research, we see new opportunities in using newborn molecular gene expression to better understand molecular pathogenesis of perinatal diseases.


[1]  Green, N.S.; Pass, K.A. Neonatal screening by DNA microarray: Spots and chips. Nat. Rev. Genet 2005, 6, 147–151.
[2]  Wilcken, B.; Wiley, V. Newborn screening. Pathology 2008, 40, 104–115.
[3]  Kammesheidt, A.; Kharrazi, M.; Graham, S.; Young, S.; Pearl, M.; Dunlop, C.; Keiles, S. Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens—Implications for newborn screening. Genet. Med 2006, 8, 557–562.
[4]  Streetly, A.; Clarke, M.; Downing, M.; Farrar, L.; Foo, Y.; Hall, K.; Kemp, H.; Newbold, J.; Walsh, P.; Yates, J.; et al. Implementation of the newborn screening programme for sickle cell disease in England: Results for 2003–2005. J. Med. Screen 2008, 15, 9–13.
[5]  Hollegaard, M.V.; Grove, J.; Thorsen, P.; Norgaard-Pedersen, B.; Hougaard, D.M. High-throughput genotyping on archived dried blood spot samples. Genet. Test. Mol. Biomark. 2009, 13, 173–179.
[6]  Hollegaard, M.V.; Thorsen, P.; Norgaard-Pedersen, B.; Hougaard, D.M. Genotyping whole-genome-amplified DNA from 3- to 25-year-old neonatal dried blood spot samples with reference to fresh genomic DNA. Electrophoresis 2009, 30, 2532–2535.
[7]  Hollegaard, M.V.; Grauholm, J.; Borglum, A.; Nyegaard, M.; Norgaard-Pedersen, B.; Orntoft, T.; Mortensen, P.B.; Wiuf, C.; Mors, O.; Didriksen, M.; et al. Genome-wide scans using archived neonatal dried blood spot samples. BMC Genomics 2009, 10, 297.
[8]  Hardin, J.; Finnell, R.H.; Wong, D.; Hogan, M.E.; Horovitz, J.; Shu, J.; Shaw, G.M. Whole genome microarray analysis, from neonatal blood cards. BMC Genet 2009, 10, 38.
[9]  Ji, H.; Li, Y.; Graham, M.; Liang, B.B.; Pilon, R.; Tyson, S.; Peters, G.; Tyler, S.; Merks, H.; Bertagnolio, S.; et al. Next-generation sequencing of dried blood spot specimens: A novel approach to HIV drug-resistance surveillance. Antivir. Ther 2011, 16, 871–878.
[10]  Winkel, B.G.; Hollegaard, M.V.; Olesen, M.S.; Svendsen, J.H.; Haunso, S.; Hougaard, D.M.; Tfelt-Hansen, J. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis. BMC Med. Genet 2011, 12, 22.
[11]  Matsubara, Y.; Ikeda, H.; Endo, H.; Narisawa, K. Dried blood spot on filter paper as a source of mRNA. Nucleic Acids Res 1992, 20, 1998.
[12]  De Crignis, E.; Re, M.C.; Cimatti, L.; Zecchi, L.; Gibellini, D. HIV-1 and HCV detection in dried blood spots by SYBR Green multiplex real-time RT-PCR. J. Virol. Methods 2010, 165, 51–56.
[13]  Lang, P.O.; Mitchell, W.A.; Govind, S.; Aspinall, R. Real time-PCR assay estimating the naive T-cell pool in whole blood and dried blood spot samples: pilot study in young adults. J. Immunol. Methods 2011, 369, 133–140.
[14]  Haak, P.T.; Busik, J.V.; Kort, E.J.; Tikhonenko, M.; Paneth, N.; Resau, J.H. Archived unfrozen neonatal blood spots are amenable to quantitative gene expression analysis. Neonatology 2009, 95, 210–216.
[15]  Khoo, S.K.; Dykema, K.; Vadlapatla, N.M.; LaHaie, D.; Valle, S.; Satterthwaite, D.; Ramirez, S.A.; Carruthers, J.A.; Haak, P.T.; Resau, J.H. Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays. Pathol. Int 2011, 61, 1–6.
[16]  Brockdorff, N.; Ashworth, A.; Kay, G.F.; Cooper, P.; Smith, S.; McCabe, V.M.; Norris, D.P.; Penny, G.D.; Patel, D.; Rastan, S. Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 1991, 351, 329–331.
[17]  Wutz, A.; Gribnau, J. X inactivation Xplained. Curr. Opin. Genet. Dev 2007, 17, 387–393.
[18]  Agulnik, A.I.; Mitchell, M.J.; Lerner, J.L.; Woods, D.R.; Bishop, C.E. A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific minor histocompatibility antigens. Hum. Mol. Genet 1994, 3, 873–878.
[19]  Kent-First, M.G.; Maffitt, M.; Muallem, A.; Brisco, P.; Shultz, J.; Ekenberg, S.; Agulnik, A.I.; Agulnik, I.; Shramm, D.; Bavister, B.; et al. Gene sequence and evolutionary conservation of human SMCY. Nat. Genet 1996, 14, 128–129.
[20]  Kim, S.J.; Dix, D.J.; Thompson, K.E.; Murrell, R.N.; Schmid, J.E.; Gallagher, J.E.; Rockett, J.C. Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood. Clin. Chem 2007, 53, 1038–1045.
[21]  Vawter, M.P.; Evans, S.; Choudary, P.; Tomita, H.; Meador-Woodruff, J.; Molnar, M.; Li, J.; Lopez, J.F.; Myers, R.; Cox, D.; et al. Gender-specific gene expression in post-mortem human brain: Localization to sex chromosomes. Neuropsychopharmacology 2004, 29, 373–384.
[22]  Watson, J.D.; Wang, S.; von Stetina, S.E.; Spencer, W.C.; Levy, S.; Dexheimer, P.J.; Kurn, N.; Heath, J.D.; Miller, D.M., III. Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system. BMC Genomics 2008, 9, 84.
[23]  Patterson, T.A.; Lobenhofer, E.K.; Fulmer-Smentek, S.B.; Collins, P.J.; Chu, T.M.; Bao, W.; Fang, H.; Kawasaki, E.S.; Hager, J.; Tikhonova, I.R.; et al. Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project. Nat. Biotechnol 2006, 24, 1140–1150.
[24]  Bolstad, B.M.; Irizarry, R.A.; Astrand, M.; Speed, T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003, 19, 185–193.
[25]  Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol 2004, 3, doi:10.2202/1544-6115.1027.
[26]  Smyth, G.K. Limma: Linear Models for Microarray Data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor; Gentleman, R., Carey, V., Dudoit, S., Irizarry, R., Huber, W., Eds.; Springer: New York, NY, USA, 2005; pp. 397–420.
[27]  Benjamini, Y.; Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. B 1995, 57, 289–300.


comments powered by Disqus