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Real-Time PCR Analysis of Metabolism-Related Genes in a Long-Lived Model of C. elegans

  • Sumino YanaseEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2138)

Abstract

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP-1 (C. elegans p53-like protein) is associated not only with the stress response, germline apoptosis, and meiotic chromosome segregation but also with longevity through the modification of energy metabolism during aging. The mitochondrial respiration-related gene sco-1 in C. elegans is orthologous to the human SCO1 gene and a target of p53/CEP-1. Using quantitative real-time polymerase chain reaction (PCR) analysis, we recently found that the expression levels of sco-1 gene were increased in wild-type C. elegans in an aging-related manner and decreased in long-lived cep-1 mutants. Here, we describe the relative quantitative strategy using a commercial real-time PCR system to detect more accurately differences in the levels of expressed genes between long-lived and wild-type C. elegans strains. To estimate the expression levels of target genes compared with wild-type using relative quantification, we used the expression levels of an endogenous control gene, such as a housekeeping gene. In addition, it is critical to normalize differences in the expression levels of the common housekeeping gene among the strains analyzed for an accurate comparison of the quantitative expression levels of target genes.

Key words

Caenorhabditis elegans p53/CEP-1 Energy metabolism Longevity TaqMan real-time PCR 

Notes

Acknowledgments

This work was supported by a special research grant from Daito Bunka University to S.Y.

References

  1. 1.
    Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314CrossRefGoogle Scholar
  2. 2.
    Matoba S, Kang J-G, Patino WD, Wragg A, Boehm M, Gavrilova O et al (2006) p53 regulates mitochondrial respiration. Science 312(5780):1650–1653CrossRefGoogle Scholar
  3. 3.
    Kondoh H, Lleonart ME, Gil J, Wang J, Degan P, Peters G et al (2005) Glycolytic enzymes can modulate cellular life span. Cancer Res 65(1):177–185PubMedGoogle Scholar
  4. 4.
    Contractor T, Harris CR (2012) p53 negatively regulates transcription of the pyruvate dehydrogenase kinase Pdk2. Cancer Res 72(2):560–567CrossRefGoogle Scholar
  5. 5.
    Derry WB, Putzke AP, Rothman JH (2001) Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science 294(5542):591–595CrossRefGoogle Scholar
  6. 6.
    Schumacher B, Hofmann K, Boulton S, Gartner A (2001) The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr Biol 11(21):1722–1727CrossRefGoogle Scholar
  7. 7.
    Arum O, Johnson TE (2007) Reduced expression of the Caenorhabditis elegans p53 ortholog cep-1 results in increased longevity. J Gerontol A Biol Sci Med Sci 62(9):951–959CrossRefGoogle Scholar
  8. 8.
    Ventura N, Rea SL, Schiavi A, Torgovnick A, Testi R, Johnson TE (2009) p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress. Aging Cell 8(4):380–393CrossRefGoogle Scholar
  9. 9.
    Yanase S, Suda H, Yasuda K, Ishii N (2017) Impaired p53/CEP-1 is associated with lifespan extension through an age-related imbalance in the energy metabolism of C. elegans. Genes Cells 22(12):1004–1010CrossRefGoogle Scholar
  10. 10.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25(4):402–408CrossRefGoogle Scholar
  11. 11.
    Lewis JA, Fleming JT (1995) Basic culture methods. In: Epstein HF, Shakes DC (eds) Caenorhabditis elegans: modern biological analysis of an organism, Methods in Cell Biology, vol 48, 1st edn. Academic Press, Cambridge, MA, USA, pp 3–29. ISBN-10: 0125641494CrossRefGoogle Scholar
  12. 12.
    Yanase S, Yasuda K, Ishii N (2019) Monitoring age-related changes in the lactate/pyruvate ratio using a colorimetric assay in a C. elegans model of increased life span. Methods Mol Biol 1916:123–132CrossRefGoogle Scholar
  13. 13.
    Yanase S, Ishii N (1999) Cloning of the oxidative stress-responsive genes in Caenorhabditis elegans. J Radiat Res 40(1):39–47CrossRefGoogle Scholar
  14. 14.
    Sambrock J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. ISBN-10: 0879693096Google Scholar
  15. 15.
    Sokolov BP, Prockop DJ (1994) A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucleic Acids Res 19:4009–4015CrossRefGoogle Scholar
  16. 16.
    Hosono R (1978) Sterilization and growth inhibition of Caenorhabditis elegans by 5-fluorodeoxyuridine. Exp Gerontol 13(5):369–374CrossRefGoogle Scholar
  17. 17.
    Mitchell DH, Stiles JW, Santelli J, Sanadi DR (1979) Synchronous growth and aging of Caenorhabditis elegans in the presence of fluorodeoxyuridine. J Gerontol 34(1):28–36CrossRefGoogle Scholar
  18. 18.
    Lee LG, Connell CR, Bloch W (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 21(16):3761–3766CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Health ScienceDaito Bunka University School of Sports and Health ScienceHigashi-MatsuyamaJapan
  2. 2.Department of Molecular Life ScienceTokai University School of MedicineIseharaJapan

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