Mutations in the mitochondrial methionyl-tRNA synthetase cause a neurodegenerative phenotype in flies and a recessive ataxia (ARSAL) in humans

PLoS Biology

Volumn 10, Issue 3, 2012, Pages

  Bayat, Vafa ^a   Thiffault, Isabelle ^b,c   Jaiswal, Manish ^a   Tétreault, Martine ^b   Donti, Taraka ^a   Sasarman, Florin ^c   Bernard, Geneviève ^b   Demers Lamarche, Julie ^b   Dicaire, Marie Josée ^b   Mathieu, Jean ^d   Vanasse, Michel ^e   Bouchard, Jean Pierre ^f   Rioux, Marie France ^g   Lourenco, Charles M ^h   Li, Zhihong ^a   Haueter, Claire ^a   Shoubridge, Eric A ^c   Graham, Brett H ^a   Brais, Bernard ^b,c,d,e   Bellen, Hugo J ^a  

^a BAYLOR COLLEGE OF MEDICINE   (United States)

^b CENTRE HOSPITALIER DE L UNIVERSITÉ DE MONTRÉAL   (Canada)

^c MCGILL UNIVERSITY   (Canada)

^d Clinique des Maladies Neuromusculaires   (Canada)

^e UNIVERSITY OF MONTREAL   (Canada)

^f LAVAL UNIVERSITY   (Canada)

^g CENTRE HOSPITALIER UNIVERSITAIRE DE SHERBROOKE   (Canada)

^h UNIVERSITY OF SÃO PAULO   (Brazil)

Author keywords

[No Author keywords available]

Indexed keywords

MARS2 PROTEIN; METHIONINE TRANSFER RNA LIGASE; MITOCHONDRIAL PROTEIN; PROTEIN; REACTIVE OXYGEN METABOLITE; UNCLASSIFIED DRUG; DROSOPHILA PROTEIN;

ARTICLE; ATAXIA; AUTOSOMAL RECESSIVE SPASTIC ATAXIA WITH LEUKOENCEPHALOPATHY; CELL PROLIFERATION; DEGENERATIVE DISEASE; DROSOPHILA; FLY; GENE; GENE DELETION; GENE DUPLICATION; GENE FUNCTION; GENE LOCUS; GENE MUTATION; GENETIC IDENTIFICATION; GENETIC SCREENING; LEIGH DISEASE; MARS2 GENE; MUTANT; NONHUMAN; NUCLEOTIDE SEQUENCE; OXIDATIVE PHOSPHORYLATION; PHENOTYPE; PHOTORECEPTOR; PROTEIN SYNTHESIS; PROTEIN UNFOLDING; SINGLE NUCLEOTIDE POLYMORPHISM; UPREGULATION; ADOLESCENT; ADULT; ANIMAL; CELL STRAIN HEK293; CHILD; ELECTRON TRANSPORT; ELECTRORETINOGRAPHY; ENZYMOLOGY; FEMALE; GENE EXPRESSION REGULATION; GENETICS; HUMAN; LEUKOENCEPHALOPATHY; LONGEVITY; MALE; METABOLISM; METHODOLOGY; MIDDLE AGED; MITOCHONDRION; MUSCLE; MUTATION; PATHOLOGY; PATHOPHYSIOLOGY; PEDIGREE; PHOTORECEPTOR CELL; PHYSIOLOGY; PRESCHOOL CHILD; RETINA; UNFOLDED PROTEIN RESPONSE;

ADOLESCENT; ADULT; ANIMALS; ATAXIA; CELL PROLIFERATION; CHILD; CHILD, PRESCHOOL; DROSOPHILA; DROSOPHILA PROTEINS; ELECTRON TRANSPORT; ELECTRORETINOGRAPHY; FEMALE; GENE EXPRESSION REGULATION, ENZYMOLOGIC; HEK293 CELLS; HUMANS; LEUKOENCEPHALOPATHIES; LONGEVITY; MALE; METHIONINE-TRNA LIGASE; MIDDLE AGED; MITOCHONDRIA; MITOCHONDRIAL PROTEINS; MUSCLES; MUTATION; NEURODEGENERATIVE DISEASES; OXIDATIVE PHOSPHORYLATION; PEDIGREE; PHENOTYPE; PHOTORECEPTOR CELLS; REACTIVE OXYGEN SPECIES; RETINA; UNFOLDED PROTEIN RESPONSE; YOUNG ADULT;

EID: 84858985882     PISSN: 15449173     EISSN: 15457885     Source Type: Journal    
DOI: 10.1371/journal.pbio.1001288     Document Type: Article

References (95)

1. Schapira A. H, (2006) Mitochondrial disease. Lancet 368: 70-82.
2. Wallace D. C, (2010) Mitochondrial DNA mutations in disease and aging. Environmental and Molecular Mutagenesis 51: 440-450.
3. Mollet J, Delahodde A, Serre V, Chretien D, Schlemmer D, et al. (2008) CABC1 gene mutations cause ubiquinone deficiency with cerebellar ataxia and seizures. Am J Hum Genet 82: 623-630.
4. Berger I, Hershkovitz E, Shaag A, Edvardson S, Saada A, et al. (2008) Mitochondrial complex I deficiency caused by a deleterious NDUFA11 mutation. Ann Neurol 63: 405-408.
5. Hudson G, Amati-Bonneau P, Blakely E. L, Stewart J. D, He L, et al. (2008) Mutation of OPA1 causes dominant optic atrophy with external ophthalmoplegia, ataxia, deafness and multiple mitochondrial DNA deletions: a novel disorder of mtDNA maintenance. Brain 131: 329-337.
6. Sarzi E, Goffart S, Serre V, Chretien D, Slama A, et al. (2007) Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion. Ann Neurol 62: 579-587.
7. Bourdon A, Minai L, Serre V, Jais J. P, Sarzi E, et al. (2007) Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 39: 776-780.
8. Amiott E. A, Lott P, Soto J, Kang P. B, McCaffery J. M, et al. (2008) Mitochondrial fusion and function in Charcot-Marie-Tooth type 2A patient fibroblasts with mitofusin 2 mutations. Exp Neurol.
9. Poole A. C, Thomas R. E, Andrews L. A, McBride H. M, Whitworth A. J, et al. (2008) The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl Acad Sci U S A 105: 1638-1643.
10. Jacobs H. T, Turnbull D. M, (2005) Nuclear genes and mitochondrial translation: a new class of genetic disease. Trends Genet 21: 312-314.
11. Mandal S, Guptan P, Owusu-Ansah E, Banerjee U, (2005) Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev Cell 9: 843-854.
12. Owusu-Ansah E, Yavari A, Mandal S, Banerjee U, (2008) Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat Genet 40: 356-361.
13. Auten R. L, Davis J. M, (2009) Oxygen toxicity and reactive oxygen species: the devil is in the details. Pediatric Research 66: 121-127.
14. Bellen H. J, Tong C, Tsuda H, (2010) 100 years of Drosophila research and its impact on vertebrate neuroscience: a history lesson for the future. Nat Rev Neurosci 11: 514-522.
15. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, et al. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392: 605-608.
16. Pesah Y, Pham T, Burgess H, Middlebrooks B, Verstreken P, et al. (2004) Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress. Development 131: 2183-2194.
17. Greene J. C, Whitworth A. J, Kuo I, Andrews L. A, Feany M. B, et al. (2003) Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc Natl Acad Sci U S A 100: 4078-4083.
18. Dodson M. W, Guo M, (2007) Pink1, Parkin, DJ-1 and mitochondrial dysfunction in Parkinson's disease. Current Opinion in Neurobiology 17: 331-337.
19. Obeso J. A, Rodriguez-Oroz M. C, Goetz C. G, Marin C, Kordower J. H, et al. (2010) Missing pieces in the Parkinson's disease puzzle. Nat Med 16: 653-661.
20. Whitworth A. J, Pallanck L. J, (2009) The PINK1/Parkin pathway: a mitochondrial quality control system? Journal of Bioenergetics and Biomembranes 41: 499-503.
21. Min K. T, Benzer S, (1997) Spongecake and eggroll: two hereditary diseases in Drosophila resemble patterns of human brain degeneration. Current Biology 7: 885-888.
22. Wang T, Montell C, (2007) Phototransduction and retinal degeneration in Drosophila. Pflugers Arch 454: 821-847.
23. Zhai R. G, Zhang F, Hiesinger P. R, Cao Y, Haueter C. M, et al. (2008) NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration. Nature 452: 887-891.
24. Zhai R. G, Cao Y, Hiesinger P. R, Zhou Y, Mehta S. Q, et al. (2006) Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity. PLoS Biol 4: e416 doi:10.1371/journal.pbio.0040416.
25. Steward O, Trimmer P. A, (1997) Genetic influences on cellular reactions to CNS injury: the reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration. J Comp Neurol 380: 70-81.
26. Mehta S. Q, Hiesinger P. R, Beronja S, Zhai R. G, Schulze K. L, et al. (2005) Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46: 219-232.
27. Hiesinger P. R, Fayyazuddin A, Mehta S. Q, Rosenmund T, Schulze K. L, et al. (2005) The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121: 607-620.
28. Thiffault I, Rioux M. F, Tetreault M, Jarry J, Loiselle L, et al. (2006) A new autosomal recessive spastic ataxia associated with frequent white matter changes maps to 2q33-34. Brain 129: 2332-2340.
29. Hotta Y, Benzer S, (1970) Genetic dissection of the Drosophila nervous system by means of mosaics. Proceedings of the National Academy of Sciences of the United States of America 67: 1156-1163.
30. Zhai R. G, Hiesinger P. R, Koh T. W, Verstreken P, Schulze K. L, et al. (2003) Mapping Drosophila mutations with molecularly defined P element insertions. Proc Natl Acad Sci U S A 100: 10860-10865.
31. Thibault S. T, Singer M. A, Miyazaki W. Y, Milash B, Dompe N. A, et al. (2004) A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet 36: 283-287.
32. Spencer A. C, Heck A, Takeuchi N, Watanabe K, Spremulli L. L, (2004) Characterization of the human mitochondrial methionyl-tRNA synthetase. Biochemistry 43: 9743-9754.
33. Brand A. H, Perrimon N, (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401-415.
34. Verstreken P, Ly C. V, Venken K. J, Koh T. W, Zhou Y, et al. (2005) Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47: 365-378.
35. Goodman J. M, (2009) Demonstrated and inferred metabolism associated with cytosolic lipid droplets. Journal of Lipid Research 50: 2148-2156.
36. Zhao Q, Wang J, Levichkin I. V, Stasinopoulos S, Ryan M. T, et al. (2002) A mitochondrial specific stress response in mammalian cells. Embo J 21: 4411-4419.
37. Haynes C. M, Petrova K, Benedetti C, Yang Y, Ron D, (2007) ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. Dev Cell 13: 467-480.
38. Yoneda T, Benedetti C, Urano F, Clark S. G, Harding H. P, et al. (2004) Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J Cell Sci 117: 4055-4066.
39. Baena-Lopez L. A, Alonso J, Rodriguez J, Santaren J. F, (2008) The Expression of heat shock protein HSP60A reveals a dynamic mitochondrial pattern in Drosophila melanogaster embryos. J Proteome Res 7: 2780-2788.
40. Tsuda H, Han S. M, Yang Y, Tong C, Lin Y. Q, et al. (2008) The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell 133: 963-977.
41. Ryoo H. D, Domingos P. M, Kang M. J, Steller H, (2007) Unfolded protein response in a Drosophila model for retinal degeneration. EMBO J 26: 242-252.
42. Daum G, Bohni P. C, Schatz G, (1982) Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257: 13028-13033.
43. Falk M. J, Rosenjack J. R, Polyak E, Suthammarak W, Chen Z, et al. (2009) Subcomplex Ilambda specifically controls integrated mitochondrial functions in Caenorhabditis elegans. PLoS One 4: e6607.
44. Sarsour E. H, Kumar M. G, Chaudhuri L, Kalen A. L, Goswami P. C, (2009) Redox control of the cell cycle in health and disease. Antioxidants & Redox Signaling 11: 2985-3011.
45. Kennedy M. C, Emptage M. H, Dreyer J. L, Beinert H, (1983) The role of iron in the activation-inactivation of aconitase. J Biol Chem 258: 11098-11105.
46. Williams M. D, Van Remmen H, Conrad C. C, Huang T. T, Epstein C. J, et al. (1998) Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem 273: 28510-28515.
47. Amer J, Atlas D, Fibach E, (2008) N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells. Biochim Biophys Acta 1780: 249-255.
48. Zhang F, Khajavi M, Connolly A. M, Towne C. F, Batish S. D, et al. (2009) The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat Genet 41: 849-853.
49. Kidd J. M, Cooper G. M, Donahue W. F, Hayden H. S, Sampas N, et al. (2008) Mapping and sequencing of structural variation from eight human genomes. Nature 453: 56-64.
50. Lee J. A, Carvalho C. M, Lupski J. R, (2007) A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 131: 1235-1247.
51. Zhang F, Carvalho C. M, Lupski J. R, (2009) Complex human chromosomal and genomic rearrangements. Trends Genet 25: 298-307.
52. Yan J, Zhang F, Brundage E, Scheuerle A, Lanpher B, et al. (2009) Genomic duplication resulting in increased copy number of genes encoding the sister chromatid cohesion complex conveys clinical consequences distinct from Cornelia de Lange. J Med Genet 46: 626-634.
53. Leary S. C, Sasarman F, (2009) Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes. Methods Mol Biol 554: 143-162.
54. Leary S. C, Sasarman F, Nishimura T, Shoubridge E. A, (2009) Human SCO2 is required for the synthesis of CO II and as a thiol-disulphide oxidoreductase for SCO1. Hum Mol Genet 18: 2230-2240.
55. Margulis L, (1975) Symbiotic theory of the origin of eukaryotic organelles; criteria for proof. Symp Soc Exp Biol pp. 21-38.
56. Wallace D. C, (2008) Mitochondria as chi. Genetics 179: 727-735.
57. Debray F. G, Lambert M, Mitchell G. A, (2008) Disorders of mitochondrial function. Current Opinion in Pediatrics 20: 471-482.
58. Scheper G. C, van der Klok T, van Andel R. J, van Berkel C. G, Sissler M, et al. (2007) Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. Nat Genet 39: 534-539.
59. Edvardson S, Shaag A, Kolesnikova O, Gomori J. M, Tarassov I, et al. (2007) Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia. Am J Hum Genet 81: 857-862.
60. Riley L. G, Cooper S, Hickey P, Rudinger-Thirion J, McKenzie M, et al. (2010) Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia-MLASA syndrome. Am J Hum Genet 87: 52-59.
61. Belostotsky R, Ben-Shalom E, Rinat C, Becker-Cohen R, Feinstein S, et al. (2011) Mutations in the Mitochondrial Seryl-tRNA Synthetase Cause Hyperuricemia, Pulmonary Hypertension, Renal Failure in Infancy and Alkalosis, HUPRA Syndrome. American Journal of Human Genetics 88: 193-200.
62. Isohanni P, Linnankivi T, Buzkova J, Lonnqvist T, Pihko H, et al. (2010) DARS2 mutations in mitochondrial leucoencephalopathy and multiple sclerosis. J Med Genet 47: 66-70.
63. Pierce S. B, Chisholm K. M, Lynch E. D, Lee M. K, Walsh T, et al. (2011) Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault syndrome. Proceedings of the National Academy of Sciences of the United States of America 108: 6543-6548.
64. Gotz A, Tyynismaa H, Euro L, Ellonen P, Hyotylainen T, et al. (2011) Exome Sequencing Identifies Mitochondrial Alanyl-tRNA Synthetase Mutations in Infantile Mitochondrial Cardiomyopathy. American Journal of Human Genetics.
65. Chamberlain S, Shaw J, Rowland A, Wallis J, South S, et al. (1988) Mapping of mutation causing Friedreich's ataxia to human chromosome 9. Nature 334: 248-250.
66. Palau F, (2001) Friedreich's ataxia and frataxin: molecular genetics, evolution and pathogenesis (Review). Int J Mol Med 7: 581-589.
67. Bartsch O, Gebauer K, Lechno S, van Esch H, Froyen G, et al. (2010) Four unrelated patients with Lubs X-linked mental retardation syndrome and different Xq28 duplications. Am J Med Genet A 152A: 305-312.
68. Cocquempot O, Brault V, Babinet C, Herault Y, (2009) Fork stalling and template switching as a mechanism for polyalanine tract expansion affecting the DYC mutant of HOXD13, a new murine model of synpolydactyly. Genetics 183: 23-30.
69. Piechota M, Korostynski M, Przewlocki R, (2010) Identification of cis-Regulatory Elements in the Mammalian Genome: The cREMaG Database. PLoS ONE 5 doi:10.1371/journal.pone.0012465.
70. Su J, Zhang Y, Lv J, Liu H, Tang X, et al. (2010) CpG_MI: a novel approach for identifying functional CpG islands in mammalian genomes. Nucleic Acids Research 38: e6.
71. Illingworth R. S, Bird A. P, (2009) CpG islands-'a rough guide'. FEBS Letters 583: 1713-1720.
72. Todd P. K, Paulson H. L, (2010) RNA-mediated neurodegeneration in repeat expansion disorders. Annals of Neurology 67: 291-300.
73. Su W. Y, Xiong H, Fang J. Y, (2010) Natural antisense transcripts regulate gene expression in an epigenetic manner. Biochemical and Biophysical Research Communications 396: 177-181.
74. Bortot B, Barbi E, Biffi S, Angelini C, Faleschini E, et al. (2009) Two novel cosegregating mutations in tRNAMet and COX III, in a patient with exercise intolerance and autoimmune polyendocrinopathy. Mitochondrion 9: 123-129.
75. Chow C. W, Thorburn D. R, (2000) Morphological correlates of mitochondrial dysfunction in children. Hum Reprod 15 (Suppl 2): 68-78.
76. Hamanaka R. B, Chandel N. S, (2011) Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes. Trends in Biochemical Sciences 35: 505-513.
77. Patten D. A, Germain M, Kelly M. A, Slack R. S, (2010) Reactive oxygen species: stuck in the middle of neurodegeneration. J Alzheimers Dis 20 (Suppl 2): S357-S367.
78. Morais V. A, De Strooper B, (2010) Mitochondria dysfunction and neurodegenerative disorders: cause or consequence. J Alzheimers Dis 20 (Suppl 2): S255-S263.
79. Parachikova A, Green K. N, Hendrix C, LaFerla F. M, (2010) Formulation of a medical food cocktail for Alzheimer's disease: beneficial effects on cognition and neuropathology in a mouse model of the disease. PLoS ONE 5: e14015 doi:10.1371/journal.pone.0014015.
80. Ouahchi K, Arita M, Kayden H, Hentati F, Ben Hamida M, et al. (1995) Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein. Nat Genet 9: 141-145.
81. Parks A. L, Cook K. R, Belvin M, Dompe N. A, Fawcett R, et al. (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat Genet 36: 288-292.
82. Mergliano J, Minden J. S, (2003) Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos. Development 130: 5779-5789.
83. Miller J. P, Holcomb J, Al-Ramahi I, de Haro M, Gafni J, et al. (2010) Matrix metalloproteinases are modifiers of huntingtin proteolysis and toxicity in Huntington's disease. Neuron 67: 199-212.
84. Venken K. J, He Y, Hoskins R. A, Bellen H. J, (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314: 1747-1751.
85. Davis R. J, Tavsanli B. C, Dittrich C, Walldorf U, Mardon G, (2003) Drosophila retinal homeobox (drx) is not required for establishment of the visual system, but is required for brain and clypeus development. Dev Biol 259: 272-287.
86. Parnas D, Haghighi A. P, Fetter R. D, Kim S. W, Goodman C. S, (2001) Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix. Neuron 32: 415-424.
87. Grenningloh G, Rehm E. J, Goodman C. S, (1991) Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell 67: 45-57.
88. Lai Z. C, Rubin G. M, (1992) Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell 70: 609-620.
89. Hofbauer A, (1991) Eine Bibliothek monoklonaler Antikorper gegen das Gehim von Drosophila melanogaster. Habilitation thesis.
90. Campbell G, Goring H, Lin T, Spana E, Andersson S, et al. (1994) RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120: 2957-2966.
91. Tayeh M. K, Chin E. L, Miller V. R, Bean L. J, Coffee B, et al. (2009) Targeted comparative genomic hybridization array for the detection of single- and multiexon gene deletions and duplications. Genet Med 11: 232-240.
92. Saillour Y, Cossee M, Leturcq F, Vasson A, Beugnet C, et al. (2008) Detection of exonic copy-number changes using a highly efficient oligonucleotide-based comparative genomic hybridization-array method. Hum Mutat 29: 1083-1090.
93. Rouleau E, Lefol C, Bourdon V, Coulet F, Noguchi T, et al. (2009) Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome. Hum Mutat 30: 867-875.
94. Morlan J, Baker J, Sinicropi D, (2009) Mutation detection by real-time PCR: a simple, robust and highly selective method. PLoS One 4: e4584 doi:10.1371/journal.pone.0004584.
95. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J, (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8: R19.