Background: State laws in the USA mandate that blood be drawn from all newborn infants to screen for health-threatening conditions. These screening assays consume only a small portion of the blood samples, which are collected on filter paper (‘Guthrie’) cards. Many states archive unused blood spots, often in unrefrigerated storage. Objectives: While individual RNA transcripts have been identified from archived neonatal blood spots, no study to date has performed quantitative analysis of archived blood spot RNA. Methods: We demonstrate that RNA can be isolated and amplified from newborn blood spots stored unfrozen for as long as 9 years, and can be analyzed by microarray and qPCR. Results: Microarray assays of archived neonatal blood spots consistently detected 3,000–4,000 expressed genes with correlations of 0.90 between replicates. Blood spot mRNA is amenable to qPCR and we detected biologically relevant expression levels of housekeeping and immune-mediating genes. Conclusions: These experiments demonstrate the feasibility of using blood spots as a source of RNA which can be analyzed using quantitative microarray and qPCR assays. The application of these methods to the analysis of widely collected biological specimens may be a valuable resource for the study of perinatal determinants of disease development.
© 2008 S. Karger AG, Basel
- Blood spot
- Real-time PCR
- Neonatal screening
- Raghuveer TS, Garg U, Graf WD: Inborn errors of metabolism in infancy and early childhood: an update. Am Fam Physician 2006;73:1981–1990.
- Green NS, Pass KA: Neonatal screening by DNA microarray: spots and chips. Nat Rev Genet 2005;6:147–151.
- Therrell BL, Johnson A, Williams D: Status of newborn screening programs in the United States. Pediatrics 2006;117:S212–S252.
- Miller AA, Sharrock KC, McDade TW: Measurement of leptin in dried blood spot samples. Am J Hum Biol 2006;18:857–860.
- Alvarez-Muñoz MT, Zaragoza-Rodríguez S, Rojas-Montes O, Palacios-Saucedo G, Vázquez-Rosales G, Gómez-Delgado A, Torres J, Muñoz O: High correlation of human immunodeficiency virus type-1 viral load measured in dried-blood spot samples and in plasma under different storage conditions. Arch Med Res 2005;36:382–386.
- Sherman GG, Stevens G, Jones SA, Horsfield P, Stevens WS: Dried blood spots improve access to HIV diagnosis and care for infants in low-resource settings. J Acquir Immune Defic Syndr 2005;38:615–617.
- Lewensohn-Fuchs I, Osterwall P, Forsgren M, Malm G: Detection of herpes simplex virus DNA in dried blood spots making a retrospective diagnosis possible. J Clin Virol 2003;26:39–48.
- Solmone M, Girardi E, Costa F, Pucillo L, Ippolito G, Capobianchi MR: Simple and reliable method for detection and genotyping of hepatitis C virus RNA in dried blood spots stored at room temperature. J Clin Microbiol 2002;40:3512–3514.
- McCabe KM, Zhang YH, Huang BL, Wagar EA, McCabe ER: Bacterial species identification after DNA amplification with a universal primer pair. Mol Genet Metab 1999;66:205–211.
- Rubin EM, Andrews KA, Kan YW: Newborn screening by DNA analysis of dried blood spots. Hum Genet 1989;82:134–136.
- Carducci C, Ellul L, Antonozzi I, Pontecorvi A: DNA elution and amplification by polymerase chain reaction from dried blood spots. Biotechniques 1992;13:735–737.
- Mackey K, Steinkamp A, Chomczynski P: DNA extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction. Mol Biotechnol 1998;9:1–5.
- Sørensen KM, Jespersgaard C, Vuust J, Hougaard D, Nørgaard-Pedersen B, Andersen PS: Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems. Genet Test 2007;11:65–71.
- Mitterer G, Bodamer O, Harwanegg C, Maurer W, Mueller MW, Schmidt WM: Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting. Genet Test 2005;9:6–13.
- Chow JC, Chen DJ, Lin CN, Chiu CY, Huang CB, Chiu PC, Lin CH, Lin SJ, Tzeng CC: Feasibility of blood spot PCR in large-scale screening of fragile X syndrome in southern Taiwan. J Formos Med Assoc 2003;102:12–16.
- Dobrowolski SF, Angeletti J, Banas RA, Naylor EW: Real-time PCR assays to detect common mutations in the biotinidase gene and application of mutational analysis to newborn screening for biotinidase deficiency. Mol Genet Metab 2003;78:100–107.
- Majumdar R, Rehana Z, Al Jumah M, Fetaini N: Spinal muscular atrophy carrier screening by multiplex polymerase chain reaction using dried blood spot on filter paper. Ann Hum Genet 2005;69:216–221.
- Olshan AF, Shaw GM, Millikan RC, Laurent C, Finnell RH: Polymorphisms in DNA repair genes as risk factors for spina bifida and orofacial clefts. Am J Med Genet A 2005;135:268–273.
- Skinner JR, Chong B, Fawkner M, Webster DR, Hegde M: Use of the newborn screening card to define cause of death in a 12-year-old diagnosed with epilepsy. J Paediatr Child Health 2004;40:651–653.
- Taub JW, Konrad MA, Ge Y, Naber JM, Scott JS, Matherly LH, Ravindranath Y: High frequency of leukemic clones in newborn screening blood samples of children with B-precursor acute lymphoblastic leukemia. Blood 2002;99:2992–2996.
- Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, Greaves MF: Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci USA 1997;94:13950–13954.
- Yagi T, Hibi S, Tabata Y, Kuriyama K, Teramura T, Hashida T, Shimizu Y, Takimoto T, Todo S, Sawada T, et al: Detection of clonotypic IGH and TCR rearrangements in the neonatal blood spots of infants and children with B-cell precursor acute lymphoblastic leukemia. Blood 2000;96:264–268.
- Wiemels JL, Xiao Z, Buffler PA, Maia AT, Ma X, Dicks BM, Smith MT, Zhang L, Feusner J, Wiencke J, et al: In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002;99:3801–3805.
- 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.
- Karlsson H, Guthenberg C, von Döbeln U, Kristenssson K: Extraction of RNA from dried blood on filter papers after long-term storage. Clin Chem 2003;49:979–981.
- Suttorp M, Ritgen M, von Neuhoff N, Schoch R, Schmitz N: Blood on filter paper as a readily available source of bcr-abl rearranged mRNA. Blood 1997;90:1713–1715.
- Pai JT, Tsai SF, Horng CJ, Chiu PC, Cheng MY, Hsiao KJ, Wuu KD: Absence of FMR-1 gene expression can be detected with RNA extracted from dried blood specimens. Hum Genet 1994;93:488–493.
- Maeno Y, Nakazawa S, Nagashima S, Sasaki J, Higo KM, Taniguchi K: 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 2003;87:295–300.
- Whitwam T, Vanbrocklin MW, Russo ME, Haak PT, Bilgili D, Resau JH, Koo HM, Holmen SL: Differential oncogenic potential of activated RAS isoforms in melanocytes. Oncogene 2007;26:4563–4570.
- Gao C, Furge K, Koeman J, Dykema K, Su Y, Cutler ML, Werts A, Haak P, Vande Woude GF: Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proc Natl Acad Sci USA 2007;104:8995–9000.
- Smyth GK, Speed T: Normalization of cDNA microarray data. Methods 2003;31:265–273.
- Ihaka R, Gentleman R: A language for data analysis and graphics. J Comput Graph Statist 1996;5:299–314.
- Applied Biosystems: User bulletin #2: ABI PRISM 7700 Sequence Detection System, 2001.
- Hembruff SL, Villeneuve DJ, Parissenti AM: The optimization of quantitative reverse transcription PCR for verification of cDNA microarray data. Anal Biochem 2005;345:237–249.
- Morey JS, Ryan JC, Van Dolah FM: Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online 2006;8:175–193.
- Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, et al: Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 2003;3:18.
- al-Naama MM, al-Naama LM, al-Sadoon TA: Glucose-6-phosphate dehydrogenase, hexokinase and pyruvate kinase activities in erythrocytes of neonates and adults in Basrah. Ann Trop Paediatr 1994;14:195–200.
- Ugarova TP, Yakubenko VP: Recognition of fibrinogen by leukocyte integrins. Ann NY Acad Sci 2001;936:368–385.
- Field LA, Jordan RM, Hadix JA, Dunn MA, Shriver CD, Ellsworth RE, Ellsworth DL: Functional identity of genes detectable in expression profiling assays following globin mRNA reduction of peripheral blood samples. Clin Biochem 2007;40:499–502.
Dr. James H. Resau
Van Andel Research Institute
333 Bostwick Ave., NE
Grand Rapids, MI 49503 (USA)
Tel. +1 616 234 5288, Fax +1 616 234 5289, E-Mail email@example.com
Received: October 16, 2007
Accepted after revision: April 22, 2008
Published online: September 18, 2008
Number of Print Pages : 7
Number of Figures : 3, Number of Tables : 1, Number of References : 39
Neonatology (Fetal and Neonatal Research)
Vol. 95, No. 3, Year 2009 (Cover Date: March 2009)
Journal Editor: Halliday H.L. (Belfast), Speer C.P. (Würzburg)
ISSN: 1661–7800 (Print), eISSN: 1661–7819 (Online)
For additional information: http://www.karger.com/NEO
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.